Increase in hepatic low-density lipoprotein receptor activity during pregnancy in Watanabe heritable hyperlipidemic rabbits; an animal model for familial hypercholesterolemia

In homozygous Watanabe heritable hyperlipidemic (WHHL) rabbits, the serum cholesterol level and serum low-density lipoprotein (LDL) level decreased from 562 +/- 76 (mean +/- S.E.) to 144 +/- 34 mg/dl and 410 +/- 56 to 90 +/- 25 mg/dl, respectively, during pregnancy, although the LDL receptor in this rabbit is genetically deficient. When Tyroxapol, which inhibits the degradation of very-low-density lipoprotein (VLDL), as well as Triton WR-1339, was injected into WHHL rabbits, the rate of the increase in serum cholesterol level in pregnant rabbits was not statistically different from that in non-pregnant rabbits. This result implied that the secretion rate of VLDL-cholesterol, the precursor of LDL-cholesterol, did not decrease during pregnancy. The amount of 125I-labeled LDL bound to LDL receptor was increased 1.8-fold in normal rabbits (from 29.3 +/- 4.3 to 52.3 +/- 4.6 ng/mg protein) and 12-fold in WHHL rabbits (from 0.5 +/- 0.2 to 6.0 +/- 0.7 ng/mg protein) during pregnancy. These results suggest that the decrease in serum cholesterol level in WHHL rabbits during pregnancy was associated with an increase in hepatic LDL receptor activity, which plays an important role in the regulation of serum cholesterol level.     

Glycosylation of LDL decreases its ability to interact with high-affinity receptors of human fibroblasts in vitro and decreases its clearance from rabbit plasma in vivo

Incubation of human LDL in vitro at 37 degrees C for 48 h with [14C]glucose at concentrations from 5 to 200 mM resulted in a glycosylated LDL, containing 0.4-20 mol of glucose incorporated per apolipoprotein B of 250 000 daltons. The extent of glucose incorporated was proportional to the time of incubation and concentration of glucose. Glycosylation of LDL abolished its uptake and degradation by the high-affinity process for LDL in normal human skin fibroblasts. 125I-labeled glycosylated LDL was bound, internalized and degraded by the fibroblasts via a nonspecific low-affinity process. The 125I-labeled glycosylated LDL and 125I-labeled LDL were taken up and degraded at similar rates in a non-saturable, low-affinity process by peritoneal macrophages isolated from mice. When 125I-labeled glycosylated LDL or 125I-labeled LDL were injected into rabbits, the glycosylated LDL had a delayed plasma clearance in comparison to the LDL. The mean fractional catabolic rates were 0.67 day-1 and 1.70 day-1 for 125I-labeled glycosylated LDL and 125I-labeled LDL, respectively. The uptake and degradation of 125I-labeled LDL by human skin fibroblasts was decreased as the concentration of free carbohydrate, glucose, sucrose or sorbitol, in the medium was increased from 10 mM to 1 M. It is speculated that pathologic levels of plasma glucose in vivo could result in a decrease in LDL uptake as a result of glycosylation of LDL. A decrease in uptake of native or modified LDL in vivo could contribute to hypercholesterolemia and its pathophysiology.     

Putative mechanisms of action of probucol on high-density lipoprotein apolipoprotein A-I and its isoproteins kinetics in rabbits

Probucol is a widely prescribed lipid-lowering agent, the major effects of which are to lower cholesterol in both low- and high-density lipoproteins (LDL and HDL, respectively). The mechanism of action of probucol on HDL apolipoprotein (apo) A-I kinetics was investigated in rabbits, with or without cholesterol feeding. 125I-labeled HDL was injected intravenously, and blood samples were taken periodically for 6 days. Kinetic parameters were calculated from the apo A-I-specific radioactivity decay curves. Fractional catabolic rate (FCR) and synthetic rate (SR) of apo A-I in rabbits fed a normal chow and normal chow with 1% probucol were similar. Apo A-I FCR of the rabbits fed 0.5% cholesterol was significantly increased but there were no changes in SR, compared to findings in the normal chow-fed group. Apo A-I FCR of the rabbits fed 1% probucol with 0.5% cholesterol (both 1 month and 2 months) was significantly increased compared to findings in rabbits fed the normal chow as well as 0.5% cholesterol diet group, while SR of apo A-I was significantly reduced in the former groups. Kinetics at 1 month after discontinuation of 1% probucol (under cholesterol feeding) showed a similar FCR of HDL-apo A-I to that of the rabbits fed 0.5% cholesterol, but the SR of apo A-I remained lower. Apo A-I isoproteins kinetics assessed by autoradiography of isoelectric focusing slab gels showed that the synthesis of proapo A-I was significantly reduced in the 1% probucol with 0.5% cholesterol administered, compared to the 0.5% cholesterol group. Thus, the action of probucol on HDL apo A-I kinetics was only prominent in case of higher serum cholesterol levels. The decreased HDL or apo A-I seen with probucol was apparently the result of an increase in FCR and a decrease in SR of HDL-apo A-I. A decreased synthesis of apo A-I remained evident even 1 month after discontinuing probucol. The action of probucol on the intracellular synthetic processes of apo A-I was revealed by the reduced synthesis of proapo A-I.     

Regulation of hepatic receptor-dependent degradation of LDL by mevinolin in rabbits with hypercholesterolemia induced by a wheat starch-casein diet

Rabbits fed a wheat starch-casein diet develop a marked hypercholesterolemia and have a slower rate of removal of rabbit 125I-labeled low density lipoproteins (LDL) from plasma. Treating rabbits with mevinolin, a highly potent competitive inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase, at a daily dose of 20 mg per animal prevents the increase in plasma and LDL cholesterol. The mevinolin effect is mediated through an increased rate of removal of rabbit 125I-labeled LDL from plasma. To study the role of mevinolin on the regulation of the hepatic LDL receptor in rabbits, the binding of 125I-labeled LDL and 125I-labeled beta-VLDL (beta-migrating very-low-density lipoproteins) to liver membranes prepared from rabbits fed the wheat starch-casein diet with or without mevinolin was investigated. Liver membranes from wheat starch-casein-fed rabbits have no demonstrable EDTA-sensitive binding activity of 125I-labeled LDL and low (37 ng/mg protein) binding activity of 125I-labeled beta-VLDL. Treatment of the wheat starch-casein fed rabbits with mevinolin results in high levels of specific EDTA-sensitive binding of 125I-labeled LDL (28.7 ng/mg protein) and 125I-labeled beta-VLDL (120 ng/mg protein). To assess the functional role of the hepatic LDL receptor in response to mevinolin, the catabolism of 125I-labeled LDL by perfused rabbit livers was studied. Perfused livers from mevinolin-treated rabbits show a 3.3-fold increase in the rate of receptor-dependent catabolism of 125I-labeled LDL (4.6% X h-1) when compared with that of livers from rabbits not treated with mevinolin (1.4% X h-1). Thus, these studies demonstrate that mevinolin prevents the increase of plasma LDL cholesterol level in rabbits fed a wheat starch-casein diet by regulating the levels of hepatic LDL-binding sites and the rate of receptor-dependent catabolism of LDL by the liver.     

Cholestyramine-induced changes in low density lipoprotein composition and metabolism. I. Studies in the guinea pig

In previous animal studies, bile acid sequestrant resins have been shown to increase the fractional catabolic rate (FCR) of a low density lipoprotein (LDL) tracer isolated from a normal donor animal and to increase hepatic LDL-receptor activity. In addition, in man, these resins are known to alter LDL composition such that low density lipoproteins are smaller, more dense, and have a decreased cholesterol:protein ratio. To determine whether metabolic consequences resulted from these changes in LDL composition, we fed cholestyramine chow (2% resin by weight) to guinea pigs, which lowered LDL cholesterol levels by 55%. LDL was isolated from control donors (C-LDL) and from cholestyramine-treated donors (CH-LDL). Compared to the C-LDL, the CH-LDL were smaller in size, depleted of cholesteryl ester and phospholipid, and had a marked decrease in their cholesterol:protein ratio. To determine whether the clearance of the altered CH-LDL was different from that of C-LDL, we labeled the two LDL preparations with 125I or 131I and simultaneously injected them into control and cholestyramine-treated guinea pigs. In 27/29 animals studied, the FCR of the CH-LDL was slower than that of C-LDL, demonstrating that the compositional changes alter the metabolism of CH-LDL. When C-LDL was used as the sole tracer in both control and treated animals, cholestyramine treatment increased the FCR by 41%; when CH-LDL was used as sole tracer, the increase in FCR on treatment was only 26%. This suggested that C-LDL was cleared more rapidly by the LDL-receptor pathway than was CH-LDL. Further support for this idea came from observations that C-LDL was degraded more readily by cultured fibroblasts and that nonenzymatic glucosylation abolished the difference in FCR between C-LDL and CH-LDL. These studies show that the effects of bile sequestration are complex and that the compositional changes produced have profound metabolic consequences. The implications of these observations for interpretation of LDL turnover studies are discussed.     

Metabolism of apoB-100 in lipoproteins separated by density gradient ultracentrifugation in normal and Watanabe heritable hyperlipidemic rabbits

We have examined the capability of a previously developed compartmental model to explain the kinetics of radioiodinated apolipoprotein (apo) B-100 in very low density lipoproteins (VLDL), intermediate density lipoproteins (IDL), and low density lipoproteins (LDL) separated by density gradient ultracentrifugation after intravenous injection of radioiodinated VLDL into New Zealand white (NZW) and Watanabe heritable hyperlipidemic (WHHL) rabbits. Our model was developed primarily from kinetics in whole blood plasma of apoB-100 in particles with and without apoE after intravenous injection of large VLDL, total VLDL, IDL, and LDL. When the initial conditions for this model were assumed to be an intravenous injection of radiolabeled VLDL, the plasma VLDL and LDL simulations for NZW rabbits and the VLDL, IDL, and LDL simulations for WHHL rabbits were found to be inconsistent with the observed density gradient data. By adding a new pathway in the VLDL portion of the model for NZW rabbits and a new compartment in VLDL for WHHL rabbits, and by assuming some cross-contamination in the density gradient ultracentrifugal separations, it was possible to bring our model, which was based upon measurements of 125I-labeled apoB-100 in whole plasma, into conformity with the data obtained by density gradient ultracentrifugation. The relatively modest changes required in the model to fit the gradient ultracentrifugation data support the suitability of our approach to the kinetic analysis of the metabolism of apoB-100 in VLDL and its conversion to IDL and LDL based upon measurements of 125I-labeled apoB-100 in whole plasma after injection of radiolabeled VLDL, IDL, and LDL. Furthermore, the differences in kinetics observed by us between data from whole plasma and data from plasma submitted to ultracentrifugal separation from the same or similar animals highlight the fact that small variations that can occur in the separation of lipoprotein classes by buoyant density can lead to confusing results.     

In Vivo Kinetics of Oxidatively Modified HDL

The kinetics of oxidatively modified high-density lipoprotein (HDL) in vivo were investigated. ¹²⁵I-labeled oxidized (Ox) I-IDL and ¹³¹I-labeled native (N) HDL were injected simultaneously into control and WHHL rabbits. The fractional catabolic rates of ¹²⁵I-labeled Ox-HDL were significantly greater than those of ¹³¹I-labeled N-HDL in both control (2.52 ± 0.36/day vs 0.94 ± 0.02/day) and WHHL rabbits (4.07/day vs 1.32/day). Oxidized HDL was catabolized faster than native HDL and was taken up primarily by the liver, spleen, and kidney.     

Human placental microvilli as a source of antigen for the preparation of a polyclonal antibody directed against the LDL receptor

Polyclonal antibodies were prepared by immunization of rabbits with partially purified LDL receptor obtained from human placental microvilli. The antiserum reacted with membranes from human placental microvilli and human fibroblasts, as assessed by immunobinding studies. It also reacted with purified LDL receptors of both origins. The antiserum markedly inhibited 125I-labeled LDL binding to cultured human fibroblasts.     

Cholesterol metabolism in fibroblasts from rabbits resistant to diet-induced hypercholesterolemia

We have previously described a colony of New Zealand White rabbits that are resistant to hypercholesterolemia when fed a cholesterol-enriched diet. The present studies used skin fibroblasts obtained from normal and hypercholesterolemia-resistant rabbits to investigate cholesterol metabolism and lipid composition in vitro. The lipid compositions of the two cell lines after incubation in either fetal calf serum or lipoprotein-deficient serum were similar. The conversion of radiolabeled acetate into sterol and phospholipids was higher in resistant fibroblasts than in normal fibroblasts. In contrast, incorporation of radiolabeled oleic acid into cholesteryl ester was significantly lower in resistant fibroblasts than in normal cells. In parallel experiments, the 3-hydroxy-3-methylglutaryl coenzyme A reductase activity was higher and acyl-coenzyme A:cholesterol acyltransferase activity was lower in resistant cells compared to normal cells. Furthermore, binding, uptake, and degradation of normal rabbit 125I-labeled LDL (low density lipoproteins) were 30% higher in resistant than in normal fibroblasts. These observations are consistent with results from previous studies of cholesterol metabolism in the liver membranes of these rabbits. The results indicate that extrahepatic cells (such as fibroblasts) from the resistant rabbit exhibit the same altered cholesterol metabolism as that found in the hepatic tissues of these rabbits. These studies suggest that the resistant rabbit may provide an in vivo and in vitro system for studying the mechanisms by which some individuals of a species can minimize the effect of dietary cholesterol on the development of hypercholesterolemia and atherosclerosis.     

Metabolic evidence for sequestration of low-density lipoprotein in abdominal aorta of normal rabbits

In rabbits, atherosclerosis develops preferentially at branch sites compared with the adjacent uniform aorta. This study investigated the hypothesis that low-density lipoprotein (LDL) is "sequestered" (present in a form that exchanges slowly with plasma LDL) in the aortas of normal rabbits and that more LDL is sequestered at branch sites. Thus 33 normal rabbits were injected with LDL labeled with (125)I-labeled tyramine cellobiose ((125)I-TC) to trace both undegraded LDL and aortic LDL degradation products. For 25 rabbits, LDL was also labeled with (131)I to trace undegraded LDL alone. The time-dependent aortic (125)I-TC and (131)I accumulation was determined from 0.6 to 120 h after injection. Compartmental modeling provided metabolic evidence for sequestration of LDL at the branch (P < 0.01) and uniform (P < 0.005) abdominal aorta. Concentrations of sequestered LDL were 109 +/- 28% higher (P < 0.0005) for branch sites. LDL mean residence time was 23.5 +/- 3.1 h for branch sites, 7.6 +/- 3.5 h longer (P < 0.05) than for the uniform abdominal aorta. Enhanced retention of higher concentrations of sequestered LDL at branch sites could account for the increased susceptibility of these aortic sites to atherosclerosis.     

Turnover of low density lipoproteins in normal and atherosclerotic rabbits

The turnover and composition of normal and hyperlipemic (h.l.) low density lipoproteins (LDL) of rabbits, were studied. They were obtained by ultracentrifugation and labeled by Bolton and Hunter method. Normal and h.l. LDL labeled with 125I were injected directly and crossed to both groups of rabbits. Normal and h.l. LDL had a different protein/lipid ratio. The analysis of fractional catabolic rate of LDL and the half-life of the phases of rapid and slow decay, show that h.l. LDL had a fractional catabolic rate that is the half of normal LDL and an increased half life of the phases of rapid and slow decay. Apparently, two factors: a) defective LDL receptor in the h.l. rabbit and b) different physico-chemical properties between normal and h.l. LDL, would be the reason for this difference. Besides, when normal and h.l. 125I LDL were injected into h.l. and normal rabbit, respectively, LDL changed according to the injected rabbit, as can be deduced from the analysis of the half life of the phase of slow decay.     

Presence of phospholipid-neutral lipid complex structures in atherosclerotic lesions as detected by a novel monoclonal antibody.

A novel monoclonal antibody (ASH1a/256C) that recognizes atherosclerotic lesions in human and Watanabe heritable hyperlipidemic (WHHL) rabbit aortae is described. When (123)I-labeled ASH1a/256C antibody is injected intravenously into WHHL rabbits, it associates specifically with fatty streaks on the aorta. The antigen recognized by the antibody is lipid, based on extraction with chloroform and methanol from WHHL rabbit tissues. The antigen, purified by high performance liquid chromatography, was shown to be phosphatidylcholine (PC), which contains unsaturated fatty acyl groups based on analyses utilizing (1)H and (13)C nuclear magnetic resonance, Fourier transfer-infrared spectrum, and mass spectrometry. The antibody did not react with other classes of phospholipids or neutral lipids when tested using an enzyme-linked immunosorbent assay. When PC was mixed with either cholesterol, cholesteryl ester, or triacylglycerol, however, the reactivity of the antibody to PC increased up to 8-fold. Homogenates of aorta tissue obtained from normal and WHHL rabbits were fractionated using sucrose density gradient ultracentrifugation in which neutral lipid droplets, cellular membranes, and proteins are separated. The phospholipid content in cellular membrane fractions from WHHL rabbits was twice as high as that of normal rabbits, and there was an enormous difference in the antigenic activity in these fractions. The content of cholesterol in the cellular membrane fraction of WHHL rabbits was approximately 50 times higher than that of normal rabbits. Addition of neutral lipids to the cellular membrane fraction of normal rabbit markedly increased the antigenic activity. Atheromatous lesions in thickened WHHL rabbit aortic intima that were rich in lipid droplets were stained positively with ASH1a/256C immunohistochemically. These results strongly suggest that PC-neutral lipid complex domains are formed in atherosclerotic lesions.     

Low-density lipoprotein catabolism in WHHL rabbits after partial ileal bypass surgery

The effect of partial ileal bypass surgery (PIB) on lipoprotein concentrations and compositions and on the catabolism of low-density lipoproteins (LDL) was studied in Watanabe heritable hyperlipidemic (WHHL) rabbits. After PIB, total serum cholesterol was 65% lower (6.22 +/- 1.58 vs. 17.24 +/- 3.22 mmol/l) and LDL cholesterol 81% lower (2.02 +/- 0.95 vs. 10.90 +/- 3.60 mmol/l) than in control WHHL rabbits; cholesteryl esters, expressed as percentage of mass, were 55% lower in the very-low and intermediate-density lipoprotein (VLDL + IDL) fractions, and 45% lower in LDL, whereas triacylglycerols were 89% higher in VLDL + IDL and 121% higher in LDL. The fractional catabolic rate (FCR) of LDL protein (apoLDL) from operated animals was 10% higher than that from controls in all animals (0.55 +/- 0.10 vs. 0.50 +/- 0.10 pools/day; P less than 0.01). The FCR of autologous apoLDL in PIB rabbits was 50% higher than that of autologous apoLDL in control rabbits (0.63 +/- 0.05 vs. 0.42 +/- 0.06 pools/day); this was not caused by induction of receptor-mediated clearance of LDL. The production rate of apoLDL after PIB in PIB rabbits was 50% lower compared to control apoLDL in controls (26.0 +/- 6.7 vs. 51.7 +/- 16.4 mg/kg per day). We conclude that PIB lowers LDL cholesterol in WHHL rabbits by a decreased production of LDL, by an increased non-specific clearance of LDL and by compositional changes, which lead to LDL particles containing less cholesterol.     

Uptake of acetylated LDL by peritoneal macrophages obtained from normal and Watanabe heritable hyperlipidemic rabbits, an animal model for familial hypercholesterolemia

Acetylated low-density lipoprotein (acetyl-LDL) stimulated the incorporation of [14C]oleate into cholesteryl [14C]oleate in peritoneal macrophages from both normal and Watanabe heritable hyperlipidemic (WHHL) rabbits. A degradation study showed that macrophages from WHHL rabbits degraded the same amount of 125I-labeled acetyl-LDL as macrophages from normal rabbits. These findings indicate that macrophages of WHHL rabbits have functional acetyl-LDL receptors.     

Defective plasma clearance of chylomicron-like lipid emulsions in Watanabe heritable hyperlipidemic rabbits

Human patients with familial hypercholesterolemia (FH) and Watanabe heritable hyperlipidemic rabbits (WHHL), while lacking normal receptors recognizing low-density lipoproteins (LDL), are said to have normal clearance of chylomicrons. In the present study, emulsions with a similar lipid composition to chylomicrons were injected intravenously in homozygous WHHL rabbits and normal control rabbits fed diet with low or high cholesterol. Radioactive labels tracing emulsion triolein and cholesteryl oleate were both removed rapidly from the bloodstream, with the removal rate of triolein always faster than that of cholesteryl oleate. This pattern was similar to the clearance of normal chylomicrons in rabbits or rats, and was consistent with the formation of remnant lipoproteins after hydrolysis of emulsion triolein by lipoprotein lipase, followed by hepatic uptake of the remnants. The removal of cholesteryl oleate was significantly slower in WHHL rabbits than in normal controls, suggesting that the absence of LDL receptor function led to impaired remnant clearance. Measured in post-heparin plasma, the activity of lipoprotein lipase was decreased in WHHL rabbits, but this was not associated with clear evidence of defective lipolysis of emulsion triolein. Apolipoprotein E did not appear to be deficient in WHHL rabbits. Plasma devoid of lipoproteins less than 1.006 g/ml from WHHL and normal control rabbits transferred similar amounts of apolipoprotein E to chylomicron-like emulsions after incubation. Impaired clearance of chylomicron remnants possibly contributes to the hypertriglyceridemia of WHHL rabbits and to accelerated atherogenesis when the function of LDL receptors is defective.     

Regulation of the uptake and degradation of beta-very low density lipoprotein in human monocyte macrophages

In normal human monocyte macrophages 125I-labeled beta-migrating very low density lipoproteins (125I-beta-VLDL), isolated from the plasma of cholesterol-fed rabbits, and 125I-human low density lipoprotein (LDL) were degraded at similar rates at protein concentrations up to 50 micrograms/ml. The high affinity degradation of 125I-labeled human LDL saturated at approximately 50 micrograms/ml; however, 125I-labeled rabbit beta-VLDL high affinity degradation saturated at 100-120 micrograms/ml. The activity of the beta-VLDL receptor was 3-fold higher than LDL receptor activity on freshly isolated normal monocyte macrophages, but with time-in-culture both receptor activities decreased and were similar after several days. The degradations of both beta-VLDL and LDL were Ca2+ sensitive, were markedly down regulated by sterols, and were up regulated by preincubation of the cells in a lipoprotein-free medium. The beta-VLDL receptor is genetically distinct from the LDL receptor as indicated by its presence on monocyte macrophages from a familial hypercholesterolemic homozygote. Human thoracic duct lymph chylomicrons as well as lipoproteins of Sf 20-5000 from fat-fed normal subjects inhibited the degradation of 125I-labeled rabbit beta-VLDL as effectively as nonradioactive rabbit beta-VLDL. We conclude: 1) the beta-VLDL receptor is genetically distinct from the LDL receptor, and 2) intestinally derived human lipoproteins are recognized by the beta-VLDL receptor on macrophages.     

Studies on the metabolism of rat serum very low density apolipoprotein.

There was a rapid transfer of radioactive peptides to other lipoprotein fractions during the first 30 min after the intravenous injection of 125I-labeled rat very low density lipoprotein (VLDL) into rats. After this initial redistribution of radioactivity, label disappeared slowly from all lipoprotein fractions. The disappearance of 125I-labeled human VLDL injected into rats was the same as that of rat VLDL. Most of the radioactivity transferred from VLDL to low density (LDL) and high density (HDL) lipoproteins was associated with two peptides, identified in these studies by polyacrylamide gel electrophoresis as zone IVa and IVb peptides (fast-migrating peptides, possibly analogous to some human C apolipoproteins), although radioactivity initially associated with zone I (analogous to human apolipoprotein B) and zone III (not characterized) was also transferred to LDL and HDL. That the transfer of label from VLDL to LDL and HDL primarily involved small molecular weight peptides was confirmed in studies using VLDL predominantly labeled in these peptides by in vitro transfer from 125I-labeled HDL. Both zone I and zone IV radioactivity was rapidly removed from VLDL during the first 5 min after injection. However, although most of the zone IV radioactivity was recovered in LDL and HDL, only 12% of the label lost from zone I of VLDL was recovered in other lipoproteins, with the remainder presumably having been cleared from the plasma compartment. We have concluded that, during catabolism of rat VLDL apoprotein, there is a rapid transfer of small molecular weight peptides to both LDL and HDL. During the catabolic process, most of the VLDL is rapidly removed from the circulation, with only a small portion being transformed into LDL molecules.     

Regulation of low density lipoprotein receptor activity by cultured human arterial smooth muscle cells.

The ability of cultured human arterial smooth muscle cells to regulate low density lipoprotein (LDL) receptor activity was tested. In contrast to human skin fibroblasts incubated with lipoprotein deficient medium under identical conditions, smooth muscle cells showed significantly reduced enhancement of 125I-labeled LDL and 125I-labeled VLDL (very low density lipoprotein) binding. Smooth muscle cells also failed to suppress LDL receptor activity during incubation with either LDL or cholesterol added to the medium, while fibroblasts shoed an active regulatory response. Thus, in comparison with the brisk LDL receptor regulation characteristic of skin fibroblasts, arterial smooth muscle cells have and attenuated capacity to regulate their LDL receptor activity. These results may be relevant to the propensity of these cells to accumulate LDL and cholesterol and form "foam cells" in the arterial wall in vivo, a process associated with atherogenesis.     

Induction of low density lipoprotein receptor synthesis by high density lipoprotein in cultures of human skin fibroblasts.

Further studies have been made of the effects of high density lipoprotein (HDL) on the surface binding, internalization and degradation of 125I-labeled low density lipoprotein (125I-labeled LDL) by cultured normal human fibroblasts. In agreement with earlier studies, during short incubations HDL inhibited the surface binding of 125I-labeled LDL. In contrast, following prolonged incubations 125I-labeled LDL binding was consistently greater in the presence of HDL. The increment in 125I-labeled LDL binding induced by HDL was: (a) associated with a decrease in cell cholesterol content; (b) inhibited by the addition of cholesterol or cycloheximide to the incubation medium; and (c) accompanied by similar increments in 125I-labeled LDL internalization and degradation. It is concluded that HDL induces the synthesis of high affinity LDL receptors in human fibroblasts by promoting the efflux of cholesterol from the cells.     

Tissue-specific mechanisms control the retention of IL-8 in lungs and skin

Chemokines are a group of structurally related peptides that promote the directed migration of leukocytes in tissue. Mechanisms controlling the retention of chemokines in tissue are not well understood. In this study we present evidence that two different mechanisms control the persistence of the CXC chemokine, IL-8, in lungs and skin. (125)I-labeled IL-8 was injected into the airspaces of the lungs and the dermis of the skin and the amount of (125)I-labeled IL-8 that remained at specified times was measured by scintillation counting. The (125)I-labeled IL-8 was cleared much more rapidly from skin than lungs, as only 2% of the (125)I-labeled IL-8 remained in skin at 4 h whereas 50% of the (125)I-labeled IL-8 remained in lungs at 4 h. Studies in neutropenic rabbits showed that neutrophils shortened the retention of (125)I-labeled IL-8 in skin but not lungs. A monomeric form of IL-8, N-methyl-leucine 25 IL-8, was not retained as long in lungs as recombinant human IL-8, indicating that dimerization of IL-8 is a mechanism that increases the local concentration and prolongs the retention of (125)I-labeled IL-8 in lungs. These observations show that the mechanisms that control the retention of IL-8 in tissue include neutrophil migration and dimerization, and that the importance of these varies in different tissues.