Visualization of unstained protein bands on polyvinylidene fluoride membranes rehydrated in Tween 20.
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Adsorption↗
Biomedical subjects
H F Hoff
Publications and source records attributed to H F Hoff.
Structure of cholesterol-containing particles accumulating in atherosclerotic lesions and the mechanisms of their derivation.
Structural and chemical modifications of plasma lipoproteins retained in atherosclerotic lesions, especially LDL, are a characteristic of atherogenesis. The major cholesterol-containing structures believed to be derived primarily from LDL are monomeric or aggregated native or modified LDL particles, cholesteryl ester droplets, liposomes rich in unesterified cholesterol, and ceroid-lipofuscin. They are suggested to be formed primarily from LDL by combinations of oxidation, hydrolysis by proteases and esterases, fusion of neutral lipid components, and covalent interactions between lipid and protein components of oxidized LDL in lysosomes. Although many of these structures appear to be refractory to removal by reverse cholesterol transport mechanisms, they may possess functional properties that still need to be elucidated.
Arteries↗
Filipin staining of lipoproteins in polyacrylamide gels: sensitivity and photobleaching of the fluorophore and its use in a double staining method.
We have developed a double staining procedure in which polyacrylamide gels are first stained with filipin to identify lipoproteins, and then with Coomassie Brilliant Blue (CBB) to identify proteins. Filipin staining when performed at 37 degrees C is both more rapid and more sensitive than previously published procedures. After only 5 min, 20 ng of low density lipoprotein (LDL) unesterified cholesterol/mm3 of band volume could be detected, and after 12 h, sensitivity reached 0.8 ng/mm3. A semilogarithmic relationship was found between the amount of LDL unesterified cholesterol applied and filipin fluorescence. Although rapid photobleaching of the fluorophore occurred during UV transillumination of these gels, such photobleaching actually resulted in maximizing of the signal:noise ratio, resulting in better definition of bands. Treatment of gels with filipin had no deleterious effects on the subsequent staining with CBB. This dual staining procedure should prove useful for studies in which both lipoproteins and proteins in plasma need to be documented in the same gel.
Cholesterol↗
Immunochemically detectable lipid-free apo(a) in plasma and in human atherosclerotic lesions.
Although Lp(a) is an independent risk factor for cardiovascular diseases in humans, the precise pathogenetic mechanisms are still unknown. We have shown that Lp(a) accumulates in human atherosclerotic lesions, and some particles undergo oxidation. Since, following agarose electrophoresis of both plaque extracts and plasma, a region close to the origin immunostained intensely for apo(a) but was lipid-free, we sought to identify whether such samples contained lipid-free apo(a), as previously reported to occur in plaque extracts. Immunochemically identifiable apo(a) was found following density-gradient ultracentrifugation both in the 1.05 < d < 1.09 and the d > 1.21 density fraction from both plasma and plaque extracts. However, because in a competitive binding RIA, displacement curves of apo(a) in plasma and the d > 1.21 were not parallel, it is premature to ascribe a relative amount of total apo(a) to this fraction. Whereas apo(a) immunoblots of SDS-PAGE under reducing conditions of the d > 1.21 fraction of a plaque extract with high apo(a) content showed high molecular weight bands consistent with apo(a) isoforms, the corresponding d > 1.21 fraction showed multiple low molecular weight bands characteristic of fragmentation. Since the d > 1.21 of arterial extracts contained all the material immunostaining for apo(a) migrating towards the cathode, characteristic of immunoglobulins (IgG), we asked whether fragments of apo(a) might have associated with human IgG both in plasma and tissue extracts, or whether our anti-apo(a) reacted with epitopes on human IgG. Immunoblotting with our anti-apo(a) of samples of plasma and plaque extracts run on agarose electrophoresis or SDS-PAGE further demonstrated intense staining of multiple bands in the molecular weight range of human IgG. Furthermore, a fraction of plasma and tissue extracts that bound to a protein G affinity column demonstrated immunostaining for apo(a) and was in the size range of IgG. Although one polyclonal anti-apo(a) provided by another laboratory showed the same findings as our antibody, two other polyclonal anti-apo(a) failed to demonstrate immunostaining of human IgG, either on agarose electrophoresis or SDS-PAGE. We speculate that the Lp(a) immunogen used to prepare our anti-apo(a) may have undergone modest oxidation, thus exposing epitopes not normally expressed on apo(a) in native Lp(a). Either antibodies to these epitopes could be recognizing apo(a) fragments, possibly released during oxidation, which are then covalently bound to IgG, or oxidation of apo(a) creates epitopes on apo(a) that are homologous with IgG, thereby leading to cross-reactivity with IgG.(ABSTRACT TRUNCATED AT 400 WORDS)
Apolipoproteins↗
Lp(a) and premature mortality during chronic hemodialysis treatment.
Lipoprotein(a) levels are approximately three to four times higher in patients with end-stage renal disease (ESRD) when compared to controls with normal renal function (H.J. Parra, H. Mezdour, C. Cachera et al., Clin. Chem. 33 (1987), 721). Hypertriglyceridemia occurs in approximately 50% of ESRD patients receiving chronic hemodialysis (HD) treatment and has been associated with an increased prevalence of cardiovascular disease (CVD) in cross-sectional studies of this subset of ESRD patients. We recently reported that HD patients with pre-existing ischemic or atherosclerotic CVD and patients with elevated Lp(a) levels had an increased risk of fatal and non-fatal clinical events attributable to CVD during a 48-month period of maintenance HD treatment. The current report describes a detailed analysis of study participants who did or did not have a history of ischemic CVD or angiographically documented severe atherosclerotic lesions prior to entry into our prospective study. Although baseline total cholesterol (TC), triglyceride (TG) and apoprotein B (apoB) levels were higher in the 36 participants with prevalent CVD than the remaining 93 study participants, total cholesterol levels were somewhat lower, while serum triglyceride levels were no different in patients who survived or experienced fatal CVD events during the period of observation on HD treatment. In contrast, Lp(a) levels were no different in participants with or without evidence of pre-existing CVD. Lp(a) was, however, an independent predictor of fatal events attributable to cardiovascular disease during the period of follow-up.
Adult↗
Inactivation of lysosomal proteases by oxidized low density lipoprotein is partially responsible for its poor degradation by mouse peritoneal macrophages.
Deficient processing of apo B in oxidized LDL (ox-LDL) by macrophage lysosomal proteases has been documented and attributed to modifications in apo B. We have investigated whether direct inactivation of lysosomal proteases by ox-LDL could also be responsible for this deficient degradation. When mouse peritoneal macrophages (MPM) were preincubated for 21 h at 37 degrees C with ox-LDL, LDL, or vortex-aggregated LDL, only ox-LDL inhibited the subsequent degradation of 125I-labeled forms of the above lipoproteins. Uptake of labeled lipoproteins was not appreciably affected by preincubation with ox-LDL, suggesting that the inhibition was at the level of lysosomal degradation. Thiol protease activity of cell extracts at pH 4.0, was reduced in MPM preincubated with ox-LDL relative to cells preincubated with LDL or medium alone. Extracts from untreated MPM, or mixtures of cathepsin B and D, showed a reduced ability to degrade 125I-LDL at pH 4.5 and reduced cathepsin B activity, after incubation with ox-LDL relative to incubation with LDL. Thus, the reduced degradation of lipoproteins in MPM pretreated with ox-LDL could be due to direct inactivation of the lysosomal protease, cathepsin B.
Animals↗
Cholesterol-specific probe for lipoproteins immobilized on nitrocellulose membranes.
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Cholesterol↗
Insoluble complex formation of lipoprotein (a) with low density lipoprotein in the presence of calcium ions.
We investigated whether apolipoprotein B-containing lipoproteins could bind to the insoluble complexes of lipoprotein (a) (Lp(a)) induced by Ca2+. Lp(a), but not low density lipoprotein (LDL), very low density lipoprotein (VLDL), or high density lipoprotein3 (HDL3) formed insoluble complexes at physiologic Ca2+ concentrations. Desialylation of Lp(a) dramatically decreased the ability of Lp(a) to aggregate, suggesting that sialic acids on Lp(a) were responsible for forming Ca2+ cross-bridges. Since a reduction of only 30% of the sialic acids on Lp(a) inhibited Ca(2+)-induced complex formation, it appears that only a small percentage of sialic acids on Lp(a) is involved in Ca(2+)-induced cross-bridging of Lp(a) particles. To determine whether other lipoproteins would complex to Lp(a) in the insoluble complexes, we mixed Lp(a) with LDL, VLDL, or HDL3 in the presence of Ca2+. Although both LDL and VLDL bound to the Lp(a) in the insoluble complexes, HDL3 not only did not bind, but it also prevented Lp(a) from forming insoluble complexes. LDL bound to Lp(a) in the insoluble complexes in a concentration-dependent manner, eventually reaching saturation at a molar ratio of 5:4 (LDL to Lp(a)). The interaction between LDL and Lp(a) appeared to be ionic, since increases in the positive charge on LDL by desialylation increased this interaction, whereas decreases in positive charge on LDL reduced this interaction. At higher Ca2+ concentrations, the binding of acetyl LDL to Lp(a) in the insoluble complexes was greater than that of LDL. Since more Ca2+ was required for concentration-dependent saturation of acetyl LDL binding, it is likely that Ca2+ cross-bridging was responsible for this binding. Thus, LDL, especially its modified forms, could contribute to the formation of insoluble complex of Lp(a) with Ca2+ in atherosclerotic lesions and help explain its preferential accumulation there.
Acetylation↗
Aggregation as well as chemical modification of LDL during oxidation is responsible for poor processing in macrophages.
Aggregation is a characteristic of extensively oxidized (ox-) LDL. We wished to determine whether this structural change contributed even more to the documented poor degradation in macrophages of ox-LDL than the chemical changes. When protein degradation of the soluble and insoluble portions of extensively ox-LDL was compared to that of acetyl LDL in mouse peritoneal macrophages (MPM), we found that the percent of internalized LDL that was degraded was lowest for the insoluble portion (insol. ox-LDL), intermediate for the soluble portion (sol. ox-LDL), and highest for the acetyl LDL, regardless of whether the binding and uptake mechanisms had been excluded, e.g., by performing appropriate pulse-chase studies. As the same order of degradation was found after long-term degradation under cell-free conditions by a mixture of cathepsin B and D, it is likely that poor degradation of ox-LDL by lysosomal proteases is partially responsible for the deficient processing of ox-LDL in MPM. However, when MPM were incubated in a pulse-chase design with LDL that was induced to aggregate by vortexing without oxidizing (vx-LDL), degradation over an 18-h interval of accumulated vx-LDL was almost as low (25%) as that of insol. ox-LDL (18%), in contrast to sol. ox-LDL (60%). Yet, in a cell-free system cathepsin degradation of vx-LDL was as efficient as that of acetyl LDL and LDL. Also, the differences in degradation between sol. and insol. ox-LDL were smaller than in MPM. Thus, it appears that alternative mechanisms to poor proteolysis of substrate were responsible for poor intracellular processing of such aggregated lipoproteins. These results suggest that, although the poorer processing of insol. ox-LDL than sol. ox-LDL may be due, in part, to more deficient proteolytic degradation, particle aggregation per se may play at least as important a role in such deficiencies. This may occur by such mechanisms as altered intracellular trafficking leading to poorer fusion in macrophages of phagosomes containing aggregated lipoproteins with lysosomes.
Acetylation↗
Co-localization of molecular mass marker proteins on western blots.
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Biomarkers↗
Structural and functional changes in LDL after modification with both 4-hydroxynonenal and malondialdehyde.
Oxidized LDL is characterized by adduct formation between apolipoprotein (apo)B-100 in the low density lipoprotein (LDL) particles and reactive aldehydes such as 4-hydroxynonenal (HNE) and malondialdehyde (MDA), which are decomposition products of lipid peroxidation. Since LDL isolated from human atherosclerotic lesions was also shown to have interacted with HNE and MDA and to have undergone oxidation, we initiated a study to structurally and functionally characterize LDL modification with both HNE and MDA and to compare these characteristics with those of LDL modified individually with HNE and MDA. Using concentrations of LDL and HNE that alone resulted in extensive particle aggregation, modification at pH 7.4 with HNE plus MDA at 20 mM and above, resulted in the prevention of particle aggregation and less aggregation of apoB-100. Increases in electrophoretic mobility and blockage of exposed lysine residues on apoB were approximately additive for LDL modified at pH 7.4 with 6 mM HNE and MDA up to 20 mM compared to LDL modified individually with HNE or individually with MDA. When 125I-labeled LDL modified at pH 7.4 with HNE and increasing amounts of MDA was incubated with J774 macrophages, the doubly modified LDL showed a linear increase in degradation and in ACAT stimulation. LDL modified with MDA alone showed first a reduction in degradation and ACAT stimulation, due to reduced recognition by the LDL receptor. This was followed by an increase in degradation when further modification with MDA was performed at pH 6.4, but no or little increase in degradation when performed at pH 7.4.(ABSTRACT TRUNCATED AT 250 WORDS)
Aldehydes↗
Partial characterization of lipoproteins containing apo[a] in human atherosclerotic lesions.
Previously we quantified the amounts of immunoreactive apo[a] found in human atherosclerotic lesions extracted sequentially with phosphate-buffered saline (PBS) and guanidine hydrochloride (GuHCl). In this study we have attempted to characterize lipoproteins containing apo[a] in such PBS and GuHCl fractions, obtained from autopsy samples, in order to eventually determine their structure-function relationships critical for evaluating the mechanisms that make them atherogenic. Apo[a] in the PBS extracts migrated slightly ahead of plasma Lp[a] on agarose electrophoresis. Although apo[a] in extracts showed the same isoforms as in plasma in SDS-PAGE, it was also highly fragmented. When a d < 1.10 g/ml ultracentrifugation fraction of the PBS extract was subjected to gel filtration, a major part of the immunoreactive apo[a] in this fraction co-isolated with plasma Lp[a]. When the Lp[a]-sized fraction was further separated by density gradient ultracentrifugation, a subpopulation was isolated containing apo[a] in the 1.06 < d < 1.08 g/ml density range that was free of lesion-derived low density lipoprotein (LDL) (A-LDL). This fraction contained immunoreactive apo[a] and apoB, had a total cholesterol to protein ratio of about 1, and demonstrated increases in fluorescence (360 ex/430 em) and conjugated dienes that were even greater than values obtained for the corresponding A-LDL sample. The void volume fraction following gel exclusion chromatography of the d < 1.10 g/ml fractions contained both apo[a] and apoB that comigrated on nondenaturing PAGE, suggesting that they were present on the same particle. Apo[a] in GuHCl extracts comigrated with plasma Lp[a] on agarose electrophoresis and contained apo[a] isoforms of similar molecular weights as those found in corresponding plasma samples. When the GuHCl extract was subjected directly to gel filtration in the presence of 6 M GuHCl, two included peaks of apo[a] immunoreactivity were present, one eluting slightly ahead of plasma Lp[a], the other slightly ahead of plasma LDL. Collectively, these data indicate that apo[a] is present in human atherosclerotic lesions in forms resembling intact but oxidized plasma Lp[a], as larger particles possibly representing Lp[a] complexed to itself or other plaque components, and as slightly smaller particles possibly representing degraded Lp[a].
Aorta↗
Oxidation of low density lipoprotein leads to particle aggregation and altered macrophage recognition.
Oxidized (ox-) low density lipoproteins (LDL) is characterized by the formation of lipid peroxides and their decomposition to reactive aldehydes which covalently link to apoB in LDL. These chemical changes are believed to be responsible for the enhanced recognition of ox-LDL by receptors on macrophages in culture. When oxidation is extensive, particle aggregation also occurs. The aim of this study was to characterize aggregation formation and how this influences the interaction of ox-LDL with macrophages in culture. When LDL was oxidized by incubating at 500 micrograms of protein/ml with 10 microM Cu2+ at 20 degrees C for up to 25 h, time-dependent increases in thiobarbituric acid reactive substances, conjugated diene content, electrophoretic mobility, and fluorescence at 360 excitation/430 emission were found. Particle aggregation increased in parallel with several parameters of oxidation and increased with increasing incubation temperatures and LDL concentrations used. When evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, apoB fragments of reproducible sizes and higher molecular weight species appeared after mild oxidation of LDL. The percent of total apoB remaining aggregated in sodium dodecyl sulfate was 50-80% at high degrees of oxidation, whereas it was far less in LDL that had been aggregated without chemical modification. This suggested that intermolecular cross-linking of apoB had occurred during oxidation of LDL at high concentrations. Degradation of ox-LDL in mouse peritoneal macrophages (MPM) increased in parallel with the degree of oxidation and with particle aggregation but reached a plateau after 12 h. Results from cross-competition studies in MPM with soluble and insoluble portions of extensively ox-LDL and with acetyl-LDL were consistent with uptake of soluble ox-LDL via both the scavenger receptor and another receptor on MPM, and uptake of the insoluble ox-LDL by an alternative mechanism.
Aldehydes↗
Lipoprotein(a) is an independent risk factor for cardiovascular disease in hemodialysis patients.
BACKGROUND: Although serum lipoprotein(a) [Lp(a)] is an independent risk factor for atherosclerosis in the general population and Lp(a) levels are increased in hemodialysis patients, an association of Lp(a) with the risk of clinical events attributed to atherosclerosis has not been established in the chronic hemodialysis patient population. We therefore determined the association between Lp(a) levels and the risk of clinical events of presumed atherosclerotic etiology in a prospective study of an outpatient hemodialysis population. METHODS AND RESULTS: Lp(a) was measured by radioimmunoassay in a baseline cardiovascular disease risk assessment in a consecutive series of 129 hemodialysis patients. The relation between baseline Lp(a) and clinical events of presumed atherosclerotic etiology was determined during 48 months of follow-up. Hemodialysis patients had a median Lp(a) concentration that was approximately four times as high as the median Lp(a) concentration in normal controls and twice as high as the levels in controls with angiographic evidence of coronary artery disease [median Lp(a), 38.4 versus 16.9 mg/dl; p less than 0.001]. Baseline Lp(a) levels were no different in participants with or with no history of a previous clinical event at the time of the baseline examination. However, baseline Lp(a) concentration (p less than 0.001) and a history of atherosclerotic clinical events (p = 0.001) were associated with clinical events during the period of follow-up. In contrast, baseline serum total cholesterol, triglyceride, high density lipoprotein cholesterol, low density lipoprotein cholesterol, age, gender, race, or duration of hemodialysis were unrelated to this risk in the prospective study. Stepwise multiple logistic regression analysis demonstrated that serum Lp(a) concentration (p = 0.001) and the presence of a previous clinical event (p = 0.004) were the only independent contributors to the risk of a clinical event during the period of follow-up. CONCLUSIONS: Lp(a) is an independent risk factor for clinical events attributed to atherosclerotic cardiovascular disease in patients receiving chronic hemodialysis treatment of end-stage renal disease.
Arteriosclerosis↗
Oxidation of LDL: role in atherogenesis.
Oxidation of LDL is proposed to accelerate atherogenesis by the following sequence of events. LDL accumulates in atherosclerotic plaques, presumably due to interaction with intimal proteoglycans. The LDL then undergoes oxidation, and aldehydic products of lipid peroxidation such as HNE or other aldehyde products derived from lipid peroxidation, induce blocking of lysine residues on apo B. This results in its recognition by the scavenger receptor on tissue macrophages at sites in which LDL concentrations are low. At sites in which the LDL concentration is high, modification with such products induces intermolecular cross-linking and particle aggregation. The aggregated, oxidized LDL particles are then phagocytosed by tissue macrophages to induce lipid loading of these cells and the formation of foam cells, a characteristic of the earliest atherosclerotic lesion. By these mechanisms oxidation of LDL accelerates atherogenesis.
Animals↗
Macrophage degradation of LDL extracted from human aortic plaques: effect of isolation conditions.
Several laboratories have recently reported on the structural and functional characteristics of an LDL fraction isolated from atherosclerotic lesions, designated A-LDL. Given the wide variety of tissue sources and isolation conditions that have been employed, we have addressed whether several procedures currently used affect the interaction of A-LDL with macrophages, and, if so, by what mechanisms. We isolated A-LDL from human aortic plaques by ultracentrifugation and gel filtration chromatography. Although some differences in the chromatographic elution profiles on gel filtration were apparent between homogenized and nonhomogenized extracts, A-LDL isolated from the same pool of plaque minces with or without homogenization showed no differences in macrophage degradation or inhibition of this degradation by excess acetyl-LDL. A-LDL isolated from plaques obtained at surgery or at autopsy less than 12 hr after death also showed no major differences in macrophage recognition, suggesting that post-mortem changes were probably not affecting cell recognition. However, A-LDL particles underwent aggregation when subjected to concentration, when stored for periods of 2 weeks or more, or when subjected to vortexing. The aggregated A-LDL was degraded more readily by macrophages than unaggregated A-LDL, and inhibition of degradation of aggregated A-LDL by excess acetyl-LDL was less than for unaggregated A-LDL. Collectively, these studies show that although post-mortem changes and tissue homogenization do not appreciably affect the interaction of A-LDL with macrophages in culture, other isolation and preparation conditions have dramatic effects which could explain some of the diversity of A-LDL metabolism reported in the literature.
Animals↗
Quantification of apo[a] and apoB in human atherosclerotic lesions.
Lipoprotein[a] or Lp[a] is a cholesterol-rich plasma lipoprotein that is associated with increased risk for cardiovascular disease. To better understand this association we determined the amount of apo[a] and apoB as possible estimates for Lp[a] and low density lipoprotein (LDL) accumulation in atherosclerotic lesions and in plasma, from patients undergoing vascular surgery, using specific radioimmunoassays for apolipoprotein[a] and apolipoprotein B. Apo[a] and apoB were operationally divided into a loosely bound fraction obtained by extracting minced samples of plaque with phosphate-buffered saline (PBS), and a tightly bound fraction obtained by extracting the residual tissue with 6 M guanidine-HCl (GuHCl). We found that 83% of all apo[a] but only 32% of all apoB in lesions was in the tightly bound fraction. When normalized for corresponding plasma levels, apo[a] accumulation in plaques was more than twice that of apoB. All fractions of tissue apo[a], loosely bound, tightly bound, and total, correlated significantly with plasma apo[a]. However, no significant correlations were found between any of the tissue fractions and plasma apoB. If all apo[a] and apoB had been associated with intact Lp[a] or LDL particles, the calculated mass of tightly bound Lp[a] would actually have exceeded that of tightly bound LDL in five cases with plasma Lp[a] levels above 5 mg apo[a] protein/dl. When PBS and GuHCl extracts of lesions were subjected to one-dimensional electrophoresis, the major band stained for lipid and immunoblotted positively for apo[a] and apoB, suggesting the presence of some intact Lp[a] in these extracts. These results suggest that Lp[a] accumulates preferentially to LDL in plaques, and that plaque apo[a] is directly associated with plasma apo[a] levels and is in a form that is less easily removable than most of the apoB. This preferential accumulation of apo[a] as a tightly bound fraction in lesions, could be responsible for the independent association of Lp[a] with cardiovascular disease in humans.
Aged↗
Macrophage uptake of low-density lipoprotein modified by 4-hydroxynonenal. An ultrastructural study.
We have documented the ultrastructural characteristics of the uptake and processing by mouse peritoneal macrophages (MPM) of low-density lipoprotein (LDL) modified with 4-hydroxynonenal (HNE), an intermediate of lipid peroxidation. This was performed as part of a larger biochemical study assessing the role of LDL oxidation in lipid loading of macrophages during atherogenesis. Gold-labeled LDL that was modified with HNE leading to particle aggregation represented the morphologic probe used. When incubated with MPM, the probe became associated with short segments of cell membrane, probably derived from blebs or from lysed cells. At 37 degrees C there was a time-dependent increase in uptake by MPM, and at 4 hours the increase paralleled the degradation by MPM of 125I-labeled HNE-LDL-cAu. Clathrin-coated pits on the cell surface were consistently associated with probe. Uptake of probe appeared to occur via phagocytosis, because pseudopods frequently surrounded probe, and cytochalasin D quantitatively prevented probe uptake. A time-dependent increase was found in the number of gold particles per unit area within vacuoles, some of which were secondary lysosomes, based on acid phosphatase-positive staining. Thus, HNE-induced aggregation of LDL during oxidation, binding of aggregates to clathrin-coated pits on MPM, and subsequent phagocytosis may represent one of the ways lipid-laden foam cells are formed in vivo.
Aldehydes↗