Absence Of Lipoprotein Lipase Research Articles

FRI092 Rationale And Design Of The Balance Study: A Randomized, Double-Blind, Placebo-Controlled, Phase 3 Study Of Olezarsen In Patients With Familial Chylomicronemia Syndrome

Abstract Disclosure: V. Alexander: Employee; Self; Ionis Pharmaceuticals Inc. E. Karwatowska-Prokopczuk: Employee; Self; Ionis Pharmaceuticals Inc. E.S. Stroes: Other; Self; Akcea Therapeutics, Ionis Pharmaceuticals Inc., Amgen Inc, Novo Nordisk, AstraZeneca, Esperion. C. Ballantyne: Speaker; Self; Ionis Pharmaceuticals Inc. H.N. Ginsberg: None. S. Xia: Employee; Self; Ionis Pharmaceuticals Inc. J. Witztum: Consulting Fee; Self; Ionis Pharmaceuticals Inc. S. Tsimikas: Employee; Self; Ionis Pharmaceuticals Inc. Background: Familial chylomicronemia syndrome (FCS) is a rare genetic cause of severe hypertriglyceridemia associated with an increased risk of acute pancreatitis (AP). Currently, there are no approved therapies for the treatment of FCS in the United States, and the absence of lipoprotein lipase (LPL) activity in FCS limits the effectiveness of current treatment options. Olezarsen, a ligand-conjugated antisense oligonucleotide (ASO), inhibits hepatic production of apolipoprotein (apo) C-III, a key regulator of LPL-mediated triglyceride (TG) lipolysis and non-LPL-mediated TG-rich lipoprotein hepatic uptake. In a phase 2 study of non-FCS patients with fasting serum TG levels of 200–500 mg/dL, olezarsen demonstrated reduction in fasting TG levels of up to 60% vs placebo (PBO). The Balance trial (NCT04568434) will evaluate the efficacy and safety of olezarsen as well as the rates of AP in patients with FCS. Methods: Balance is a phase 3, randomized, double-blind, PBO-controlled trial. Key inclusion criteria are genetic confirmation of FCS, fasting TG ≥ 880 mg/dL, and possible history of pancreatitis. Key exclusion criteria include clinically significant abnormalities in medical history such as major surgery within 3 mos of screening and active pancreatitis within 4 wks of screening. Patients will maintain ≤ 20 g daily fat intake and most will receive lipid-lowering medications per local standard of care. Results: Eligible patients (N=66) were enrolled at 29 sites (across 11 countries) and randomized 2:1 to receive one of two doses of olezarsen or PBO over a 53-wk treatment period, followed by post-treatment evaluation or entry into an open-label extension study. Enrollment was monitored to ensure that ≥ 65% of patients had history of pancreatitis within 10 yrs. Reported baseline parameters are preliminary. Of enrolled patients, mean age is 45±13.4 yrs, 58% of the population are female, and mean body mass index is 24.0 kg/m2; 83% had a history of hospitalization for AP, or severe abdominal pain episode, or symptoms suggestive of pancreatitis, with a mean of 2.4 episodes over the last 5 yrs and 5.1 over the last 10 yrs. Median fasting TG level is 2334.5 mg/dL. The primary efficacy endpoint of the study is percent change in fasting TG from baseline (BL) at 6 mos with olezarsen compared to PBO. Secondary endpoints (olezarsen vs PBO) include percent change in fasting TG from BL at 12 mos, achievement of ≥ 40% or ≥ 70% fasting TG reduction at 6 mos, patient proportions ≤ 500 mg/dL or ≤ 750 mg/dL at 6 mos, and adjudicated AP event rates. Exploratory assessments include patient-reported symptoms such as abdominal pain and health-related quality of life. Safety and tolerability assessments include adverse events and laboratory tests. Conclusion: Balance is designed to determine whether the apoC-III ASO olezarsen, added to standard of care, safely reduces TG levels and the rate of AP in patients with FCS. Presentation: Friday, June 16, 2023

Familial Chylomicronaemia: A Neonate with Milky White Blood

Familial Chylomicronaemia is a rare autosomal recessive disease of lipoprotein metabolism characterized by deficiency or absence of lipoprotein lipase (LPL) or its co-factor apoC-II which causes severe elevation of triglyceride and chylomicron resulting in lipaemic plasma, recurrent attacks of acute pancreatitis, eruptive xanthomas, hepato-splenomegaly and lipaemiaretinalis. We report a case of term female neonate with lipaemic plasma, lipemia retinalis, markedly elevated triglyceride level which is consistent with diagnosis of Familial Chylomicronaemia.
 Keywords: Familial Chylomicronemia, lipemic plasma, triglyceride level, Lipemia retinalis

Lipoprotein Lipase Modulates Bone Marrow Myeloid Cell Proliferation by Affecting Colony‐Stimulating Factor Levels and Recruitment

Although inflammation is the body's basic response to ameliorate injury or infection, tissue macrophage‐mediated pro‐inflammatory responses promote metabolic diseases, such as atherosclerosis. Lipoprotein lipase (LpL) is the rate‐limiting enzyme that hydrolyzes circulating lipoprotein triglycerides (TG) to release free fatty acids. We have reported that loss of LpL expression reduced numbers of aortic macrophages and bone marrow (BM) myelopoiesis. Reconstitution of LpL‐expressing BM was able to replenish aortic macrophage density. Our current studies are directed toward understanding the molecular regulation of LpL in orchestrating lipoprotein metabolism and immune cell biology. Our data show that reduced aortic macrophage levels were associated with reduced blood white cells and leukocyte subsets, including macrophage precursor cells‐ monocytes. BM‐derived monocyte differentiation to macrophages was markedly reduced by 40% in the absence of LpL (p<0.05). We speculate that defective lipid metabolism induced by LpL deficiency can contribute to decreased BM myelopoiesis. In transgenic LpL knockout mice that are rescued from neonatal death by muscle‐specific LpL expression (MCKL0 mice), loss of LpL reduced BM monocyte/macrophage precursor (BMMP) cell TG uptake and accumulation by 22% and 50% (p<0.05), respectively. TG utilization was much lower (~40%) in MCKL0 BMMP cells compared to wild type (WT) BMMP cells. LpL deficiency significantly decreased BM expression of both macrophage‐colony‐stimulating factor (M‐CSF) and its receptor (M‐CSF‐R), required for myeloid cell proliferation and differentiation (p<0.05). LpL‐mediated lipolysis products (e.g., oleic acid) were able to stimulate expression of M‐CSF and M‐CSF‐R by > 35% (p<0.05) in WT BMMP cells. In addition, LpL and heparin, which releases LpL from the cell surface, modulated binding of M‐CSF to M‐CSF‐R in the immunobinding assay (p<0.05). We hypothesize that LpL also anchors M‐CSF to its receptor and triggers a series of downstream signaling pathways to stimulate monocyte differentiation to macrophages. We thus propose that LpL, in addition to being a lipolytic enzyme, also influences peripheral and BM immune cell responses. These studies provide valuable insight into the regulation of adverse inflammatory responses and have therapeutic applications for various blood cell diseases.

Loss of Lipoprotein Lipase Expression Reduces Circulating Monocytes and Decreases Bone Marrow Myeloid Proliferation

Tissue macrophages (Mq) perpetuate pro‐inflammatory responses that promote metabolic diseases and atherogenesis. Lipoprotein lipase (LpL) is synthesized by Mq and smooth muscle cells and is also associated with endothelial cells. We have reported that dietary saturated fatty acids increased LpL with substantial amounts of LpL being Mq‐associated and heparin‐releasable in the arterial wall. We studied whether LpL expression altered the circulating levels and tissue content of monocytes (MO)/Mq. In transgenic LpL knockout mice rescued by muscle‐specific LpL expression (MCKL0 mice), loss of LpL in the arterial wall reduced the number of aortic Mq by >30% and this reduction was reversed by reconstitution with LpL‐expressing bone marrow (BM) (p<0.05). The decrease in aortic Mq in MCKL0 mice was associated with ~40% reduction in blood total leukocytes, neutrophils, and total and pro‐inflammatory MO (Gr‐1hi) (p<0.05). LpL deficiency significantly decreased BM Mq‐associated markers (F4/80 and TNF‐α), and led to a marked reduction (~50‐70%) in BM expression of transcription factors (PU.1 and C/EBPα) and colony‐stimulating factors (CSFs) and their receptors (CSF‐Rs) required for myeloid cell proliferation and differentiation (p<0.05). BM‐derived MO differentiation to Mq was markedly reduced by 40% in the absence of LpL (p<0.05). Our studies indicate that LpL plays a key role in mediating blood MO levels and MO differentiation to Mq, in part, through affecting CSFs and CSF‐Rs levels.Grant Funding Source: NIH‐HL40404 (RJD); HL450945 (IJG); T32‐DK007674 /HL007343 (CLC)

Lipoprotein Lipase Is a Novel Amyloid β (Aβ)-binding Protein That Promotes Glycosaminoglycan-dependent Cellular Uptake of Aβ in Astrocytes

Lipoprotein lipase (LPL) is a member of a lipase family known to hydrolyze triglyceride molecules in plasma lipoprotein particles. LPL also plays a role in the binding of lipoprotein particles to cell-surface molecules, including sulfated glycosaminoglycans (GAGs). LPL is predominantly expressed in adipose and muscle but is also highly expressed in the brain where its specific roles are unknown. It has been shown that LPL is colocalized with senile plaques in Alzheimer disease (AD) brains, and its mutations are associated with the severity of AD pathophysiological features. In this study, we identified a novel function of LPL; that is, LPL binds to amyloid β protein (Aβ) and promotes cell-surface association and uptake of Aβ in mouse primary astrocytes. The internalized Aβ was degraded within 12 h, mainly in a lysosomal pathway. We also found that sulfated GAGs were involved in the LPL-mediated cellular uptake of Aβ. Apolipoprotein E was dispensable in the LPL-mediated uptake of Aβ. Our findings indicate that LPL is a novel Aβ-binding protein promoting cellular uptake and subsequent degradation of Aβ.

Discovery Of A Novel Anti-atherogenic Function Of Lipoprotein Lipase In Human Coronary Endothelial Cells

Acute physical inactivity profoundly suppresses extracellular LPL activity below normal control levels and exercise rapidly raises LPL activity above normal. Lipoprotein lipase (LPL) is a candidate gene associated with reduced risk for cardiovascular disease in epidemiological studies. Exercise putatively improves cardiovascular health by effects on the endothelium. Given these possibilities, there is much to be gained by determining if there are mechanism(s) that LPL activity directly influences endothelial cells, especially over an acute time frame. PURPOSE We tested the hypothesis that LPL activity regulates the expression of thrombospondin-1 (TSP-1) in endothelial cells. TSP-1 is a secretory protein that is expressed at high levels in atherosclerotic vessels and stimulates several pro-atherogenic processes in the endothelium and underlying smooth muscle cells. METHODS The effect of changing LPL activity over an acute time span (1–24 hrs) was determined in primary endothelial cell cultures obtained from healthy humans and pigs. LPL and VLDL were derived from healthy donors. Fatty acid free albumin was included to maintain free fatty acids at a physiological concentration in the media. Northern and Western blots were used for mRNA and protein measurements, respectively. RESULTS There was only a modest trend for TSP-1 expression to increase after adding moderate concentrations of VLDL (0.12 mM triglyceride) in the absence of LPL. In contrast, LPL combined with VLDL suppressed TSP-1 expression several fold (P<0.01). LPL caused a gradual decrease in TSP-1 expression in both porcine and human endothelial cells, leading to a 60–95% decrease after 12–24 hrs (P<0.01). In addition to the lowering of TSP-1, LPL activity was also necessary for maintaining a high level of eNOS protein concentration in cells challenged with an inflammatory stress caused by TNFα. CONCLUSION These studies provide a rationale for the novel hypothesis that exercise may restore/preserve a healthy endothelial phenotype and attenuate CAD in large part by LPL dependent-transcriptional responses in endothelial cells.

Triacylglycerol-rich lipoprotein–gene interactions in endothelial cells

Lipoproteins such as LDL (low-density lipoprotein) and oxidized LDL have potentially adverse effects on endothelial cells due to their ability to activate pro-inflammatory pathways regulated via the transcription factor NF-kappaB (nuclear factor kappaB). Triacylglycerol-rich lipoproteins (the chylomicrons, very-low-density lipoprotein and their respective remnant particles) have also been implicated in the induction of a pro-inflammatory phenotype and up-regulation of adhesion molecule expression. Although early studies supported the proposal that LPL (lipoprotein lipase)-mediated hydrolysis of TRLs (triglyceride-rich lipoproteins) at the endothelium could activate the NFkappaB pathway, more recent studies provide evidence of pro- and anti-inflammatory responses when cells are exposed to fatty acids or TRL particles. A large number of genes are up- and down-regulated when cells are exposed to TRL, with the net effect reflecting receptor- and nonreceptor-mediated pathways that are activated or inhibited depending on fatty acid type, the lipid and apolipoprotein composition of the TRL and the presence or absence of LPL. Early concepts of TRL particles as essentially pro-inflammatory stimuli to the endothelium provide an overly simplistic view of their impact on the vascular compartment.

Lipoprotein metabolism in a coculture system with eel skeletal muscle cells and hepatocytes

: Eel muscle contains lipid as much as 15–20% of wet weight of muscle. However, it is uncertain whether lipid is synthesized in muscle or transported from another tissue such as liver. We prepared isolated muscle cells to investigate the synthetic ability of lipid by muscle cell itself and studied lipoprotein metabolism in coculture with eel hepatocytes. Isolated eel muscle cells were prepared by collagenase digestion of skeletal muscle and their length and width were approximately 2.2 mm and 5–20 µm, respectively. The length corresponded to that of myotome between myocommata. Cultured muscle cells synthesized lipid and protein from 14C-acetate, but the ability of lipid synthesis by muscle cells was below 1/100 that of eel hepatocytes. In the absence of lipoprotein lipase (LPL), the highest component among 14C-lipid changed from 14C-phospholipid in muscle cells cultured alone to 14C-triacylglycerol (14C-TG) in cocultured muscle cells. These results suggest that cocultured muscle cells incorporate the very low-density lipoprotein (VLDL)-like lipoprotein secreted by cocultured hepatocytes, of which the main lipid is 14C-TG. In the presence of LPL in the coculture system, 14C-lipid in muscle cells increased 1.6-fold during 48 h incubation compared to that without LPL. 14C-Free fatty acids to total 14C-lipid in muscle cells increased from 1.1 to 32.6% by addition of 6 U/mL of LPL. Furthermore, the secreted VLDL-like lipoprotein was decreased and a gel filtration column separated formed high-density lipoprotein (HDL)-like lipoprotein. These results suggest that 14C-free fatty acids produced by LPL digestion of 14C-VLDL-like lipoprotein were transported to muscle cells. We concluded that there are two metabolisms of the secreted lipoprotein dependent or independent on LPL.

Defective uptake of triglyceride-associated fatty acids in adipose tissue causes the SREBP-1c-mediated induction of lipogenesis

Lipoprotein lipase (LPL) is the only known enzyme in the capillary endothelium of peripheral tissues that hydrolizes plasma triglycerides and provides fatty acids (FAs) for their subsequent tissue uptake. Previously, we demonstrated that mice that express LPL exclusively in muscle develop essentially normal fat mass despite the absence of LPL and the deprivation of nutritionally derived FAs in adipose tissue (AT). Using this mouse model, we now investigated the metabolic response to LPL deficiency in AT that enables maintenance of normal AT mass. We show that the rate of FA production was 1.8-fold higher in LPL-deficient AT than in control AT. The levels of mRNA and enzymatic activities of important enzymes involved in FA and triglyceride biosynthesis were induced concomitantly. Increased plasma glucose clearing and (14)C-deoxyglucose uptake into LPL-deficient mouse fat pads indicated that glucose provided the carbon source for lipid synthesis. Leptin expression was decreased in LPL-deficient AT. Finally, the induction of de novo FA synthesis in LPL-deficient AT was associated with increased expression and processing of sterol regulatory element binding protein 1 (SREBP-1), together with an increase in INSIG-1 expression. These results suggest that in the absence of LPL in AT, lipogenesis is activated through increased SREBP-1 expression and processing triggered by decreased availability of nutrition-derived FAs, elevated insulin, and low leptin levels.

Induced mutant mouse lines that express lipoprotein lipase in cardiac muscle, but not in skeletal muscle and adipose tissue, have normal plasma triglyceride and high-density lipoprotein-cholesterol levels.

The tissue-specific expression of lipoprotein lipase (LPL) in adipose tissue (AT), skeletal muscle (SM), and cardiac muscle (CM) is rate-limiting for the uptake of triglyceride (TG)-derived free fatty acids and decisive in the regulation of energy balance and lipoprotein metabolism. To investigate the tissue-specific metabolic effects of LPL, three independent transgenic mouse lines were established that expressed a human LPL (hLPL) minigene predominantly in CM. Through cross-breeding with heterozygous LPL knockout mice, animals were generated that produced hLPL mRNA and enzyme activity in CM but lacked the enzyme in SM and AT because of the absence of the endogenous mouse LPL gene (L0-hLPL). LPL activity in CM and postheparin plasma of L0-hLPL mice was reduced by 34% and 60%, respectively, compared with control mice. This reduced LPL expression was sufficient to rescue LPL knockout mice from neonatal death. L0-hLPL animals developed normally with regard to body weight and body-mass composition. Plasma TG levels in L0-hLPL animals were increased up to 10-fold during the suckling period but normalized after weaning and decreased in adult animals. L0-hLPL mice had normal plasma high-density lipoprotein (HDL)-cholesterol levels, indicating that LPL expression in CM alone was sufficient to allow for normal HDL production. The absence of LPL in SM and AT did not cause detectable morphological or histopathological changes in these tissues. However, the lipid composition in AT and SM exhibited a marked decrease in polyunsaturated fatty acids. From this genetic model of LPL deficiency in SM and AT, it can be concluded that CM-specific LPL expression is a major determinant in the regulation of plasma TG and HDL-cholesterol levels.

Binding of beta-VLDL to heparan sulfate proteoglycans requires lipoprotein lipase, whereas ApoE only modulates binding affinity.

The binding of beta-VLDL to heparan sulfate proteoglycans (HSPG) has been reported to be stimulated by both apoE and lipoprotein lipase (LPL). In the present study we investigated the effect of the isoform and the amount of apoE per particle, as well as the role of LPL on the binding of beta-VLDL to HSPG. Therefore, we isolated beta-VLDL from transgenic mice, expressing either APOE*2(Arg158-->Cys) or APOE*3-Leiden (E2-VLDL and E3Leiden-VLDL, respectively), as well as from apoE-deficient mice containing no apoE at all (Enull-VLDL). In the absence of LPL, the binding affinity and maximal binding capacity of all beta-VLDL samples for HSPG-coated microtiter plates was very low. Addition of LPL to this cell-free system resulted in a 12- to 55-fold increase in the binding affinity and a 7- to 15-fold increase in the maximal binding capacity (Bmax). In the presence of LPL, the association constant (Ka) tended to increase in the order Enull-VLDL<E2-VLDL<E3Leiden-VLDL, whereas Bmax increased in the reverse order: E3Leiden-VLDL approximately E2-VLDL<Enull-VLDL. Addition of LPL resulted in a marked stimulation of both Ka and Bmax for binding of beta-VLDL samples to J774 cells similar to that found for the binding to HSPG-LPL complexes. Our results indicate that both Ka and Bmax for binding of beta-VLDL to HSPG are increased more than 1 order of magnitude on addition of LPL. In addition, for the binding of beta-VLDL to HSPG-LPL complexes, the presence of apoE is not a prerequisite, but results in an increased binding affinity, depending on the apoE isoform used.

Interaction of lipoproteins with heparan sulfate proteoglycans and with lipoprotein lipase. Studies by surface plasmon resonance technique.

Interaction of different classes of lipoproteins with heparan sulfate, heparin, and lipoprotein lipase was studied by a surface plasmon resonance based technique on a BIAcore. The proteoglycans were covalently attached to sensor chips as previously described [Lookene, A., Chevreuil, O., Ostergaard, P., & Olivecrona, G. (1996) Biochemistry 35, 12155-12163]. Binding of all lipoproteins, except for beta-VLDL, to endothelial heparan sulfate was low. Binding of chylomicrons (from rat lymph) and of human VLDL was much increased by the presence of lipoprotein lipase. With human LDL, binding was low in the absence of lipase or at low lipase concentrations. For efficient binding, 2-4 lipase dimers per LDL particle were necessary, indicating cooperativity in the interaction. In contrast, HDL did not bind under any conditions. Heparin had higher binding capacity for lipoproteins than heparan sulfate. This was due to a higher number of binding sites on the heparin chains. Binding of LDL, VLDL, and chylomicrons to heparan sulfate-covered surfaces, both in the presence and in the absence of lipoprotein lipase, was characterized by high values for association rate constants (10(4)-10(5) M(-1) s(-1)) and low values for dissociation rate constants (10(-4)-10(-5) M(-1) s(-1)). In some experiments, rabbit beta-VLDL were directly immobilized to the sensor chips. Binding of lipoprotein lipase to these surfaces was characterized by a very high association rate constant (10(6) M(-1) s(-1)). The dissociation of triacylglycerol-rich lipoproteins was more rapid with catalytically active lipase than with active site-inhibited lipase. It was also markedly increased in the presence of free heparin, suggesting fast exchange kinetics at the surface. Based on that, we propose that lipoproteins are relatively mobile at heparan sulfate covered surfaces. Our study emphasizes the important role of lipoprotein lipase, or molecules with similar properties (apolipoprotein E, hepatic lipase), as mediators for binding of lipoproteins to proteoglycans. It also demonstrates the great potential for the use of biosensors for studies of lipoprotein interactions.

Lipoprotein lipase-enhanced binding of human triglyceride-rich lipoproteins to heparan sulfate: modulation by apolipoprotein E and apolipoprotein C

The objective of this study was to investigate whether compositional variation in apolipoprotein (apo) content of triglyceride-rich lipoproteins (TRLP) modulates binding of heparan sulfate proteoglycans (HSPG). Human TRLP was enriched with apoE and apoCs and the ability to bind biotin-conjugated heparan sulfate (b-HS) was studied in the presence or absence of heat-inactivated lipoprotein lipase (LPL). TRLP, associated with LPL, showed an increased capacity to bind b-HS compared with TRLP alone. Low density lipoproteins (LDL) bound both b-HS and LPL with a higher affinity than TRLP. ApoE enrichment of TRLP resulted in an enhanced binding of b-HS. Increased binding of b-HS to TRLP by the combination of apoE enrichment and LPL addition was found to be complementary, not affecting their individual binding capacity. TRLP enrichment with apoC led to the formation of an apoC-rich, apoE-poor particle; this alteration by itself did not change the ability to bind b-HS. ApoC enrichment of TRLP resulted in a reduced capacity to bind LPL and therefore a subsequently reduced capacity to bind b-HS, compared with control TRLP associated with LPL. Competition studies revealed that b-HS binding to TRLP was fully displaceable by lactoferrin but barely by heparan sulfate, dermatan sulfate, or chondroitin-4-sulfate. Using TRLP coated to microtiter wells and associated with LPL, the b-HS displacement patterns were comparable to those obtained with coated LDL in the presence or absence of LPL. The cell-free system that was used enabled us to identify the functions of apoC and apoE in the binding of TRLP to LPL and HSPG. Both LPL and apoE increased the ability of TRLP to bind HSPG. The apoC content of TRLP regulated the docking of TRLP to LPL. ApoC enrichment reduced the affinity or capacity of TRLP to LPL binding, and this has relevance for the lipolytic cascade.

Homozygosity for two point mutations in the lipoprotein lipase (LPL) gene in a patient with familial LPL deficiency: LPL(Asp9–>Asn, Tyr262–>His)

Familial lipoprotein lipase (LPL) deficiency is an inherited disorder of lipoprotein metabolism characterized by hypertriglyceridemia and recurrent episodes of abdominal pain and pancreatitis. We have studied the genetic basis of LPL deficiency in a 62-year-old black male with undetectable pre- and post-heparin plasma LPL mass and activity, DNA sequence analysis of the patient's LPL cDNA and gene as well as digestion with Bcl I and Asu I revealed that the proband is a homozygote for two separate gene defects. One mutation changed a G to an A, resulting in the conversion of amino acid 9 of the mature protein, aspartic acid (GAC), to asparagine (AAC). The second substitution, a C for a T, replaced tyrosine (TAC) at residue 262 with histidine (CAC). Northern blot analysis of monocyte-derived macrophage RNA demonstrated the presence of LPL mRNA of approximately normal size and quantity when compared to control. Expression of both mutations separately (pCMV-9 and pCMV-262) or in combination (pCMV-9+262) in human embryonal kidney-293 cells demonstrated that LPL-9 had approximately 80% the specific activity of wild type LPL, but LPL-262 and LPL-9+262 had no enzymic activity, thus establishing the functional significance of the LPL-262 defect. Despite an absolute deficiency of LPL mass and activity demonstrated by analysis of patient post-heparin plasma, in vitro expression of both LPL mutants was normal, suggesting that the absence of LPL in patient post-heparin plasma was a result of altered in vivo processing. Analysis of the heparin binding properties of the mutant enzymes by heparin-Sepharose affinity chromatography indicated that most of the LPL-262 mass was present in an inactive peak, which like the normal LPL monomer, eluted at 0.8 M NaCl. Thus, the Tyr262 --> His mutation may alter the stability of the LPL dimer, leading to the formation of inactive LPL-262 monomer which exhibits reduced heparin affinity. Based on these results, we propose that, in vivo, enhanced formation of LPL-9+262 monomer leads to abnormal binding of the mutant lipase to endothelial glycosaminoglycans ultimately resulting in enhanced catabolism of the mutant enzyme and lower enzyme mass in post-heparin plasma.

Lipoprotein lipase-mediated uptake of lipoprotein in human fibroblasts: evidence for an LDL receptor-independent internalization pathway

Lipoprotein lipase (LPL), a key enzyme in lipoprotein triglyceride metabolism, produces a marked increase in the retention and uptake of all classes of lipoproteins by cultured cells. It was previously shown that two different receptors are involved in mediating the LPL effects: heparan sulfate proteoglycans (HSPG) and the low density lipoprotein (LDL) receptor-related protein/alpha 2 macroglobulin receptor (LRP). By immunofluorescence we show here that cell surface-bound LPL displays a pattern that corresponds to the previously described distribution of cell surface HSPG. No evident relation to the distribution of bound activated alpha 2-macroglobulin (alpha 2M*) or to LRP was observed. By immunoelectron microscopy we found that after 30 min at 37 degrees C most of the detected alpha 2M* (70% of the total gold particles) was inside the cells and associated with endosomal vesicles. However, at the same time, 76% of the LPL remained at the cell surface, suggesting that, LPL is internalized by a slow endocytic process. Binding of triglyceride-rich lipoproteins (TRL) or LDL together with LPL led to a spectacular increase in bound lipoproteins, which completely colocalized with LPL. After incubation at 37 degrees C, LPL and 1,1'-dioctadecyl-3,3,3,'3'-tetramethylindocarbocyanine (DiI)-TRL formed large clusters on the cell surface. Immunofluorescene and quantitative immunoelectron microscopy provided evidence of co-internalization of LPL and apoE-containing TRL by a slow endocytic process. In the absence of LPL, the fibroblasts rapidly internalized DiI-LDL and showed fluorescence in central, lysosome-like vesicles. In contrast, when LPL was present, internalization of DiI-LDL involved small, widely distributed vesicles. This pattern slowly changed to one consisting of large perinuclear vesicles. LDL receptor-deficient fibroblasts internalized DiI-LDL, either with or without LPL, into small widely distributed vesicles and no central vesicles were seen. Chloroquine-treated normal fibroblasts internalized DiI-LDL in a pattern similar to that of receptor-deficient fibroblasts. Taken together our results suggest an alternative receptor-independent endocytosis pathway for LDL. This pathway is potentiated by LPL and is characterized by a slow uptake involving small vesicles that gradually reach lysosomes. We suggest that, through its interaction with HSPG, LPL provides high capacity binding sites for lipoproteins and a independent internalization pathway.

Oxidation of Low Density Lipoproteins Greatly Enhances Their Association with Lipoprotein Lipase Anchored to Endothelial Cell Matrix

Native and oxidized low density lipoprotein retention within arterial wall endothelial cell matrix (ECM) is an early event in the pathogenesis of atherosclerosis. Previously we showed lipoprotein lipase (LPL) addition to ECM enhanced the retention of apoB-containing lipoproteins. In the present studies we examined whether the oxidation of low density lipoprotein (LDL) increases its retention by LPL-containing ECM. Except where noted, 125I-labeled moderately oxidized LDL (ModOxLDL) was prepared by long term storage of 125I-LDL. Without LPL, 125I-ModOxLDL matrix binding was low and nonsaturable. LPL preanchored to ECM resulted in 125I-ModOxLDL binding that was saturable and 20-fold greater than in the absence of LPL, with an association constant equal to 2.6 nM. Copper-oxidized LDL (Cu-OxLDL) was able to compete with 125I-ModOxLDL, whereas a 60-fold native LDL excess had no effect. Reconstituted apolipoprotein B from Cu-OxLDL also reduced 125I-ModOxLDL to LPL, whereas liposomes derived from the lipid extract of Cu-OxLDL had no effect on binding. These data suggest that the increased binding of oxidized LDL to LPL-ECM may be due to the exposure of novel apoB binding sites and not an oxidized lipid moiety. 125I-ModOxLDL binding was also not affected by either preincubation with a 300-fold molar excess of apoE-poor HDL or an 340-fold molar excess of Cu-Ox-HDL. In contrast, a 4-fold apoE-rich HDL excess (based on protein) totally inhibited 125I-ModOxLDL matrix retention. Positively charged peptides of polyarginine mimicked the effect of apoE-rich HDL in reducing the 125I-ModOxLDL retention; however, polylysine had no effect. We postulate that the oxidation of LDL may be a mechanism that enhances LDL retention by the ECM-bound LPL and that the protective effects of apoE-containing HDL may in part be due to its ability to block the retention of oxidized LDL in vivo.

Cellular differences in lipoprotein lipase-mediated uptake of low density lipoproteins.

Lipoprotein lipase (LPL) increases the cellular uptake and degradation of LDL by fibroblasts and macrophages via a heparin-sensitive process. The roles of the LDL receptor, LDL receptor-related protein (LRP), and proteoglycans in this process were studied. In up-regulated human fibroblasts, LPL increased degradation of 125I-low density lipoprotein (LDL) (5 micrograms/ml) only 30% during a 6-h incubation at 37 degrees C. Monoclonal antibody 47 (which interacts with the receptor binding region of apoB) decreased LDL degradation 93% in the absence of LPL, but did not reduce the LPL-mediated increase in degradation. In contrast, addition of the 39-kDa receptor-associated protein (RAP) caused a 43% decrease in the LPL-dependent LDL degradation in non-up-regulated fibroblasts. Monoclonal antibody 47 did not decrease LDL degradation by THP-1 macrophages and RAP caused a < 13% decrease in LPL-mediated LDL degradation. LPL also increased the association of acetyl LDL with the surface of the macrophages but did not increase acetyl LDL degradation. The kinetics of LPL-mediated LDL metabolism in macrophages was then compared with that in fibroblasts. The half-lives of cell surface LDL and LPL during a subsequent 37 degrees C incubation were approximately 1 h in THP-1 cells versus 6 h in fibroblasts. In addition, 50% of the 125I-LDL and 30% of the 125I-LPL were degraded within 3 h. After metabolic labeling of THP-1 proteoglycans with 35SO4, > 30% of pericellular heparan sulfate was lost between 2-4 h of the chase period. Therefore, some of the LPL-mediated LDL degradation in the THP-1 cells could be accounted for by internalization of cell surface proteoglycans. We conclude that LRP, but not the LDL receptor, is involved in LPL-mediated degradation of LDL in fibroblasts. This process is much more rapid in THP-1 cells and in addition to LRP may involve other receptors and internalization of proteoglycans.

Relationship of VLDL receptor and LPL in metabolism of VLDL by macrophage

Macrophages were incubated with 125I-VLDL for 5 h in presence or absence of lipoprotein lipase (LPL) inhibitor, benzene boronic acid (BBA). Both the uptake and degradation of 125I-VLDL by macrophages were saturable, and the uptake and degradation curves were virtually identical. When macrophages were incubated with 125I-VLDL for 10 h in presence of BBA, the uptake and degradation of 125I-VLDL were still saturable. However, in absence of BBA, the uptake and degradation were no longer saturable. The results suggest that with macrophages incubated with VLDL for a shorter period, VLDL was taken up predominantly via receptor pathway, with a longer period of incubation, LPL played a striking role in uptake of VLDL.

The role of the endothelium in myocardial lipoprotein dynamics.

This overview is presented, in the main, to summarize the following areas of myocardial lipoprotein metabolism: 1. The nature and extent of the cardiac endothelium. 2. The interactions between the endothelium and chylomicrons, very low, low and high density lipoproteins in the presence and absence of lipoprotein lipase. 3. The importance of the endothelial lipoprotein lipase and the mechanisms involved in the enzymes' sequestration at that site. 4. The physiological role of lipoprotein lipase in the provision of oxidizable fuel for the heart.

Effect of lipoprotein lipase and hepatic triglyceride lipase activity on the distribution of apolipoprotein E among the plasma lipoproteins

The independent roles of human lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) in determining the distribution of apolipoprotein E (apo E) among the plasma lipoproteins has been studied in vitro. In one series of three studies, postheparin plasma (10%) was incubated for 2 h with autologous plasma and the changes in the lipoprotein association of apo E after lipase exposure were determined after lipoprotein fractionation on 4% agarose columns. Specificity for LPL or HTGL was achieved by inhibition with goat anti-human HTGL or with 1 M NaCl, respectively. In another study, LPL and HTGL were partially purified from human postheparin plasma. The independent effects of these enzymes on the lipoprotein association of apo E were then examined after incubation of plasma in the absence or presence of one or both lipases. Data from both types of in vitro study showed that LPL-mediated triglyceride hydrolysis in the absence of HTGL activity was accompanied by a loss of apo E from triglyceride-rich lipoproteins, a gain or no change in the apo E-containing lipoproteins the size of intermediate density lipoproteins (IDL) and inconsistent changes in the apo E mass associated with high density lipoproteins (HDL). HTGL activity, on the other hand, in the absence of LPL, resulted in a redistribution of apo E from lipoproteins the size of IDL and a gain by those of HDL size. These studies thus support previous in vivo studies which pointed toward a specific role for HTGL in the processing of apo E containing IDL.