Intercellular adhesion molecule-1-deficient mice have antibody responses but impaired leukocyte recruitment.

The role of intercellular adhesion molecule-1 (ICAM-1) in murine lung inflammation was examined in vivo. Ovalbumin (Ova)-sensitized and -challenged ICAM-1-deficient (KO) mice had decreased accumulation of leukocytes in the bronchoalveolar lavage fluid compared with wild-type (WT) mice. Lung tissue inflammation was also attenuated. Ova immunization and challenge produced equivalent plasma levels of Ova-specific immunoglobulin (Ig) G1 and higher concentrations of IgE in KO versus WT mice. Ova-dependent induction of cytokines in vitro, as measured by enzyme-linked immunosorbent assay, was impaired in splenocytes from KO mice compared with the comparable release of interleukin (IL)-5 and IL-10 from anti-CD3-stimulated WT and KO splenocytes. Methacholine-induced increases in trapped gas in lungs of Ova-sensitized and -challenged WT mice were greater than those of KO mice. The activation of lung tissue nuclear factor-kappa B was diminished in KO mice after Ova provocation. This suggests that ICAM-1 was important for activation of the inflammatory cascade leading to the recruitment of leukocytes but was not critical for the generation of antibody responses in vivo.

Biotransformation of lifibrol (U-83860) to mixed glyceride metabolites by rat and human hepatocytes in primary culture.

Lifibrol (U-83860), K12.148) is a lipid-lowering drug that has the potential to accumulate in the liver and induce hepatic peroxisome proliferation. To investigate the identity and potential human relevance of persistent lifibrol-related residues in rat liver, rat and human hepatocyte primary cultures were treated with 30 microM of [14C]lifibrol. After a steady uptake for 24 hr, cellular levels of radioactivity became stable for the next 24-48 hr. A nonradioactive lifibrol chase caused an efflux of intracellular radioactivity. Cellular autoradiography revealed the association of radioactivity with small lipid drops at 6 hr exposure and with large lipid drops at 24 hr exposure. HPLC analysis of media revealed that lifibrol acyl glucuronide and a t-butylcarboxylic acid metabolite (U-94613) were the major metabolites of rat and human hepatocytes, respectively. Using an HPLC method suitable for nonpolar metabolites, the analysis of rat and human cell extracts revealed a broad band of multiple, radioactive peaks that had a similar retention and UV spectrum to a synthetic standard of lifibrol cholesterol ester. Folch extracts of liver from rats treated with [14C]lifibrol or unlabeled lifibrol and [14C]acetate had a unique radioactive TLC band that had similar HPLC retention to hepatocyte residues. The group of nonpolar peaks from the hepatocytes was purified by HPLC. Conversion of the lifibrol sec-hydroxy group to a nicotinate ester afforded particle beam-electron impact mass spectra of the cholesterol ester standard and hepatocyte residues. The derivatized rat hepatocyte residue did not contain detectable lifibrol cholesterol ester (M+.816), but did contain molecular ion clusters corresponding to a mixed triglyceride of lifibrol and two fatty acids. Lifibrol-specific product ions of molecular ion clusters centered at M+.1021, 1047, and 1073 were observed at m/z 448, 430, and 310. The major lifibrol-containing triglycerides had a fatty acid composition of C16-C20 with 0-6 unsaturations.

NF-kappa B is activated during acute inflammation in vivo in association with elevated endothelial cell adhesion molecule gene expression and leukocyte recruitment.

Leukocytes accumulate at sites of inflammation in response to the induced expression of endothelial cell adhesion molecules. The nuclear transcription factor kappa B (NF-kappa B) plays a critical role in the cytokine-induced expression of these genes in cultured endothelium. We examined the relationship between NF-kappa B activation and endothelial cell adhesion molecule gene expression in vivo during the initiation of acute inflammation. Nuclear NF-kappa B DNA-binding activity was rapidly increased within lung and heart tissues of rats administered endotoxin, consistent with the translocation of NF-kappa B complexes from the cytoplasm to the nucleus. This NF-kappa B was composed of p50 and p65 subunits, and could bind NF-kappa B elements in the E-selectin promoter. NF-kappa B activation was maximal within 30 min and persisted for at least 3 hr after endotoxin treatment. NF-kappa B activation preceded the transcriptional activation of the P-selectin, E-selectin, VCAM-1, and ICAM-1 genes. In the lung, increased expression of P-selectin and ICAM-1 protein was detected immunohistochemically. These molecular events were temporally associated with the sequestration of leukocytes and the development of pulmonary inflammation. NF-kappa B activation is therefore an early event in the initiation of acute inflammation in vivo. This molecular pathway may be of consequence in the pathogenesis of acute inflammatory disease.

Long-term effects of Aroclor 1254 (PCBs) on plasma lipid and carnitine concentrations in rhesus monkey.

Sixty-seven female rhesus monkeys (Macaca mulatta) were orally dosed daily for 152 weeks with 0, 5, 20, 40, and 80 micrograms Aroclor 1254 (PCB)/kg body wt. Blood polychlorinated biphenyl (PCB) concentrations were highly positively correlated (r = 0.92, P < 0.001) with doses of PCB administered. A comprehensive analysis of plasma lipids/lipoproteins revealed a PCB-associated increase in plasma triglycerides and decreases in plasma total cholesterol, high-density lipoprotein cholesterol (HDL-chol), very-low plus low-density lipoprotein cholesterol (VLDL+LDL-chol), and total carnitine (which is involved in fatty acid metabolism). All of the lipid/lipoprotein changes were significantly (P < or = 0.05) correlated with blood PCB concentration. These data, obtained after 152 weeks of continuous daily exposure of a primate model to PCB support a causal relationship between plasma lipid changes and PCB intake. Previously, causality has been refuted on the premise that the commonly observed elevation of triglycerides with increasing concentration of blood PCB is a reflection, not of PCB dose, but of the partitioning of PCB between tissues (adipose) and blood in proportion to the blood lipid present. The mechanism of the plasma lipid changes was not investigated in this study but the altered lipid/lipoprotein pattern is discussed with respect to known cardiovascular risk profiles.

Action of the new hypolipidemic agent lifibrol (K12.148) on lipid homeostasis in normal rats: plasma lipids, hepatic sterologenesis, and the fate of injected [14C]acetate.

Lifibrol, a new hypocholesterolemic agent with activity in humans, was examined in normal rats for its short-term and long-term effects on lipid homeostasis. Cholesterol (Chol) synthesis inhibition by lifibrol was demonstrated in vitro in liver minces from normal rats by following [1-14C]acetate ([14C]Ac) and DL-[2-14C]mevalonate ([14C]-MVA) incorporation into [14C]Chol. When administered at 50 mg/kg/d, lifibrol reduced plasma total Chol and triglycerides (TG) (P < 0.001) within 24 h. The Chol reduction was largely a result of reduction of low density and very low density lipoprotein cholesterol (LDL + VLDL-chol) and a smaller decrease in high density lipoprotein cholesterol (HDL-chol). After 10 d, however, a rebound effect emerged, and after 41 d, plasma Chol, LDL + VLDL-chol, and HDL-chol were restored. In contrast, plasma TG remained at reduced levels (P < 0.01). The rebound is attributed to counter-regulation of hepatic sterologenesis that was assessed both ex vivo and in vivo. The ex vivo incorporation of [14C]MVA and [14C]octanoate into [14C]Chol and total digitonin-precipitable [14C]sterols ([14C]DPS) in liver minces was increased 2-and 6-fold, respectively, in rats treated 6 d at 50 mg/kg. Similarly, in vivo incorporation of intraperitoneally injected [14C]Ac into hepatic [14C]DPS (2 h post-injection) was increased 2- to 5-fold at 50 mg/kg, and evidence for increased sterologenesis in nonhepatic tissue was also obtained. The increased hepatic sterologenesis, evident within 48 h, persisted out to 41 d of treatment by which time increases (P < 0.002) in hepatic Chol and carcass total sterols were observed.(ABSTRACT TRUNCATED AT 250 WORDS)

Effect of the lipid-lowering drug lifibrol on lipid metabolism in rat macrophages and in atherosclerotic arteries from swine and WHHL rabbits, in vitro. Implications in atherogenesis.

The effects of lifibrol on lipid metabolism in rat macrophages and swine and rabbit aortae were investigated. Resident peritoneal macrophages isolated from rats pretreated with lifibrol (50 mg/kg/7 days) showed a decreased capacity to synthesize cholesteryl esters from labeled precursors ([1-14C]oleate and [4-14C]cholesterol). Macrophages isolated similarly from non-treated rats demonstrated the ability to take up [14C]lifibrol, in vitro. Modification of lipid metabolism in atherosclerotic aortae from swine and Watanabe heritable hyperlipidemic (WHHL) rabbits was also observed when the tissues were incubated in vitro in the presence of exogenous lifibrol. Concentrations of lifibrol of up to 50 micrograms/mL in the incubations selectively reduced the formation of cholesteryl esters from [1-14C]acetate by 60-75%, whereas higher concentrations (100 micrograms/mL) resulted in a generalized inhibition of lipid biosynthesis of about 50% and of cholesteryl ester formation by up to 90%. The ability of lifibrol to directly affect these targets (i.e. macrophages and arterial tissue) has implications that extend beyond its confirmed plasma cholesterol-lowering activity since early stages of the atherogenic process involve an overall increase in arterial lipid synthesis and cholesteryl ester accumulation by monocyte-macrophages that infiltrate the vessel wall from blood.

Temporal evaluation of fatty acid-binding protein (FABP) activity in association with the development of atherosclerosis in the rabbit.

1. The relationship between atherosclerosis development and changes in arterial fatty acid binding protein (FABP) activity was investigated in the aortas of New Zealand rabbits which were fed an atherogenic diet containing 1% cholesterol and 3% peanut oil for 16 weeks. 2. At 4-week intervals, FABP activity, cholesterol and microsomal acylCoA:cholesterol acyltransferase (ACAT) activity were determined in aortic tissue and serum cholesterol was measured; age-matched normal rabbits served as control comparators. 3. Serum cholesterol increased from 35 mg/dl in the normal rabbits to 2290 mg/dl in the 16-week cholesterol-fed rabbits. 4. The microsomal fraction isolated from cholesterol-fed rabbit aortas exhibited a progressive elevation in ACAT activity as time on the diet increased. By 12-16 weeks, ACAT activity had increased approximately 10-fold relative to normal activity. 5. Arterial cholesterol content of the cholesterol-fed animals increased from less than 2 mg/g wet weight to greater than 10 mg/g wet weight at 12 and 16 weeks. In contrast, arterial FABP activity gradually decreased with time on the cholesterol diet; a significant decrease (P less than 0.05) was observed at 16 weeks, where palmitoyl CoA binding was decreased from 61.0 to 36.3 pmol/mg protein. 6. In the cholesterol-fed rabbits, total arterial cholesterol and ACAT activity showed a significant (P less than 0.05) inverse correlation to FABP activity with correlation coefficients of -0.93 and -0.95, respectively. 7. Additionally, FABP activity increased significantly (P less than 0.05) in the 16-week normal rabbit as compared to the 4-week normal rabbit, suggesting an age-dependent interaction.

L-carnitine administration and withdrawal affect plasma and hepatic carnitine concentrations, plasma lipid and lipoprotein composition, and in vitro hepatic lipogenesis from labeled mevalonate and oleate in normal rabbits.

Carnitine was administered to normal rabbits to investigate the possible effects of pharmacologic doses on various aspects of normal lipid and lipoprotein metabolism. Carnitine concentrations were measured in the plasma and liver of normal rabbits that received L-carnitine orally [40 mg/(kg.d)] for 21 d and after withdrawal from the carnitine supplement for 21 d. Plasma lipids, plasma lipoprotein composition and in vitro hepatic lipid biosynthesis from [2-14C]mevalonate and [1-14C]oleate were also measured. Threefold elevations in plasma carnitine with carnitine treatment were essentially reversed after 48 h of carnitine withdrawal, but elevated hepatic carnitine accumulation (twofold) persisted for 21 d, suggesting that the accumulated carnitine constituted a pool that is only slowly miscible with plasma. The rabbits withdrawn from L-carnitine for 21 d experienced a 35% decrease in plasma cholesterol, a 50% decrease in VLDL cholesterol, and an increase in the protein content of HDL and of intermediate density lipoprotein + LDL. Additionally, the proportion of [14C]oleate incorporated into hepatic phospholipids increased 35% at the expense of triglyceride and the ratio of hepatic [14C]cholesterol to [14C]squalene derived from [14C]mevalonate increased over twofold following carnitine withdrawal. These studies provide evidence that normal lipid homeostasis can be altered by supplemental carnitine and that the perturbations are reflected by changes in plasma lipids and lipoproteins and in the proportions of the hepatic lipids synthesized.

U-73482: a novel ACAT inhibitor that elevates HDL-cholesterol, lowers plasma triglyceride and facilitates hepatic cholesterol mobilization in the rat.

U-73482, a novel acylCoA:cholesterol acyltransferase (ACAT) inhibitor with systemic activity, has been evaluated for its effects on a variety of lipid metabolic parameters in the rat. The compound inhibits ACAT in vitro in cultured Fu5AH rat hepatoma cells and demonstrates systemic activity through inhibition of hepatic ACAT in rats receiving the drug orally. U-73482 also lowers plasma triglycerides at 40 mg/kg per day in the rat and elevates high density lipoprotein cholesterol (HDL-chol) in a dose-related fashion over the range of daily intakes of 0-40 mg/kg in the rat. Elevations in HDL-chol are followed by elevations in total plasma cholesterol in normal rats but the compound exerts hypocholesterolemic activity in cholesterol-fed rats and promotes clearance of stored hepatic sterol in rats pretreated with a hypercholesterolemic diet and then changed over to normal chow. The triglyceride-lowering and HDL-chol elevating effects of U-73482 coupled with its ability to promote tissue sterol clearance and block the hypercholesterolemic effects of dietary cholesterol in animals, suggests that the compound has potential as a therapeutic agent for treatment of lipid disorders in man.

Evidence for an altered lipid metabolic state in circulating blood monocytes under conditions of hyperlipemia in swine and its implications in arterial lipid metabolism.

Circulating blood monocytes were isolated from normal and hypercholesterolemic swine, and the monocyte lipid compositions and lipid biosynthesis profiles were assessed. The data indicate that monocytes freshly isolated from hyperlipemic swine have increased phospholipid and cholesterol contents and have increased biosynthetic capability for synthesizing phospholipids, triglycerides, and cholesteryl esters, but not cholesterol. The profile of the stimulated lipid synthesis capability is similar to that of the swine aortic intima undergoing atherogenic change. These studies indicate that circulating blood monocytes in hyperlipemic swine, which are known to give rise to intimal foam cells in the early fatty streak lesion, can contribute to altered vessel lipid metabolism without a requirement for in situ modification by wall factors.

Synthesis and hypocholesterolemic activity of 6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-diones, novel inhibitors of acylCoA:cholesterol O-acyltransferase.

A novel series of 6,7-dihydro-4H-pyrazolo[1,5-a]pyrrolo[3,4-d]pyrimidine-5,8-dione inhibitors of the enzyme acyl-CoA:cholesterol O-acyltransferase is described. A number of these derivatives were found to be potent modulators of serum lipoprotein levels in cholesterol-fed rats. Further evaluation of one of the most effective analogues confirmed that it was significantly blocking the absorption of cholesterol from the gut.

Arterial lipid biochemistry in the spontaneously hyperlipidemic Zucker rat and its similarity to early atherogenesis.

In the present studies, arterial lipid metabolism was evaluated in the spontaneously hyperlipidemic obese Zucker rat (fa/fa), the lean Zucker rat (Fa/-), and the Sprague-Dawley (SD) rat. Mean serum cholesterol levels in the obese Zucker, lean Zucker and SD rats were 216 +/- 18 mg/dl, 145 +/- 14 mg/dl and 84 +/- 5 mg/dl, respectively. Arterial cholesterol content was in the same rank order as plasma cholesterol and ranged from a mean of 2.23 +/- 0.10 mg/gm wet wt. in the obese rats to 1.36 +/- 0.04 mg/gm wet wt. in the SD rats. The increased arterial sterol in the obese rats was associated with increased lipid metabolism activity. The in vitro incorporation of [14C]oleate into arterial cholesteryl esters was increased 3-4-fold (P less than 0.01) and incorporation into phospholipids and triglycerides was also elevated (P less than 0.001 and P less than 0.01, respectively). The arterial sterol content and arterial lipid metabolism pattern observed in obese Zucker rat aortas are similar to those found in vessels of other species undergoing atherogenic change.

Production of an antibody to arterial fatty acid-binding protein (FABP) using a synthetic peptide as the antigen.

1. A procedure is described for the preparation of an antibody to arterial FABP using a synthetic peptide as an antigen. In order to locate a highly conserved region located on the outer surface of FABP, computer analysis of primary and secondary structures of several proteins from the FABP family was undertaken and a 24 amino acid sequence beginning at the fifth position from the N-terminus of rat heart FABP was chosen. 2. The synthetic peptide consisted of eight replications of the 24 amino acid sequence individually attached to the alpha and epsilon amino groups of each terminal lysine on an octalysine branched peptide. 3. Antibody to the synthetic antigen was raised in New Zealand rabbits. Western analysis was conducted and detection was accomplished by using goat-anti-rabbit second antibody conjugated to alkaline phosphatase. 4. The antibody produced from the previously described peptide, recognized purified rat heart FABP and demonstrated a high positive correlation (r = 0.96) when known concentrations of purified hFABP were plotted against densitometric measurement of the bands. 5. Additionally, the antibody recognized FABP from the 104,000 g supernates of rat atrial and arterial tissue fractionated by a Sephadex G-75 column. 6. Therefore, the antibody produced from this particular protocol employing a synthetic peptide can be utilized qualitatively and quantitatively in the analysis of the heart and arterial FABP content.

Lipid metabolism in the inflammatory lesion of a mouse model of delayed type hypersensitivity. I. Increased acylCoA:cholesterol acyltransferase activity as an integral component of the inflammatory response.

Lipid metabolism was studied in a normal model of delayed type hypersensitivity to methylated bovine serum albumin (MBSA). Following initial sensitization to a single injection of MBSA, MBSA-soaked millipore filter disks (10 mm diam) were implanted in subcutaneous pockets and the course of development and the resolution of the inflammatory lesions were followed biochemically for 35 days. Of particular interest were our observations that the activity of the cholesterol esterifying enzyme, which is attributed to acyl-CoA:cholesterol acyltransferase (ACAT; EC 2.3.1.26) was increased up to 38-fold in the developing inflammatory lesion and represented the single most dramatic alteration in lipid metabolism to occur. As the lesions began to show histological evidence of resolving (between 21 and 35 days), ACAT activity declined toward basal levels. The data suggest the possibility that the ACAT reaction is an important component of the inflammatory response and, as such, offers the possibility of a novel approach to controlling the inflammatory process through ACAT inhibition.

Proliferative and lipid metabolism response to balloon angioplasty in canine renal arteries.

The mechanisms responsible for reocclusion after percutaneous transluminal angioplasty are still poorly understood. The effects of angioplasty on arterial morphology, deoxyribonucleic acid (DNA) synthesis (3H-thymidine incorporation) and lipid metabolism (14C-oleate incorporation) were studied in renal arteries of 24 male mongrel dogs. Balloon-dilated (identified by Evans blue dye accumulation) and adjacent normal arterial segments were collected 90 min and 2, 5 and 14 days after the procedure. The immediate vascular response was endothelial cell denudation and platelet accumulation. Two weeks after angioplasty, healing of the luminal surface by "endothelial-like" cells, mild smooth muscle cell proliferation and an angiogenic response with capillary growth into the media were observed. DNA synthesis was increased in balloon-dilated segments at day 5 compared with adjacent nonballoon-dilated artery. This increase in DNA synthesis persisted in the 2 week postangioplasty segments. Additionally, angioplasty produced both quantitative and qualitative changes in arterial lipid synthesis. The most dramatic change was an increase in sterol esterification that was apparent as early as 90 min after angioplasty; the change persisted through day 5 but diminished toward baseline by day 14. Angioplasty-induced alterations of arterial metabolism parallel aspects of the atherogenic process and may be involved in the pathogenesis of postangioplasty reocclusion, particularly in the presence of additional risk factors, such as hyperlipemia.

Arterial fatty acid-binding protein activity associated with dietarily-induced and spontaneously occurring atherosclerosis in the rabbit (Oryctolagus cuniculus).

1. Adult WHHL rabbits, or New Zealand rabbits fed either a stock chow diet or a high cholesterol diet were evaluated to assess the relationship between the development of aortic atherosclerosis and arterial FABP activity. 2. Aortic FABP activity was significantly (P less than 0.05) lower in atherosclerotic New Zealand aortas (0.039 +/- 0.008 nmol palmitoyl CoA bound/mg soluble prot) which had developed macroscopic lesions on 80% of the aortic surface as compared to lesion-free New Zealand aortas (0.053 +/- 0.002 nmol palmitoyl CoA bound/mg soluble prot). 3. In spontaneously hyperlipidemic rabbit (WHHL) aortas, FABP activity (0.023 +/- 0.004 nmol palmitoyl CoA bound/mg soluble prot) was significantly lower (P less than 0.05) than in either the normal or atherosclerotic New Zealand aortas. 4. To our knowledge, this study is the first to report a change in arterial FABP with the atherogenic process.

Comparison of lipid accumulation and metabolism in carrageenan-induced granulomas to aorta and blood monocytes of normal and cholesterol-fed rabbits.

New Zealand rabbits (six each) were either maintained on a standard chow diet (ND) or the chow diet supplemented with cholesterol/peanut oil (HD) for 2 weeks. After 2 weeks, each animal had 15 ml of a 1% carrageenan gel injected subcutaneously into the midabdominal area. Rabbits were maintained on the diets for an additional 4 weeks. At sacrifice, blood was collected both for serum and for monocyte isolation and granulomas and aorta were excised. Tissues were assayed for lipid composition and lipid metabolism. Electron and light microscopies were performed on granuloma tissue. Granulomas from ND animals did not stain with oil red O. Granulomas from HD animals had homogenous oil red O staining indicating lipid accumulation. Granulomas from both ND and HD animals consisted of macrophages. Macrophages from ND rabbits accumulated follicular carrageenan but not lipid, while HD macrophages had the appearance of foam cells. Granuloma lipid content and metabolism closely paralleled the aorta and blood monocytes. The HD tissue had increased acylCoA:cholesterol acetyltransferase (ACAT) activity and lipid composition changes reflective of the atherosclerotic process. ND granulomas had no elevation of lipid content or ACAT. The carrageenan-induced granulomas provide a useful model for studying the biochemical and morphologic changes characteristic of arteries undergoing atherogenic change.

A study of atherosclerotic lesion development in the injured pulmonary arteries of dogs with induced hyperlipemia.

Studies of experimental atherosclerosis in the dog demonstrate that many months at plasma cholesterol concentrations greater than 750 mg/dl are required to produce lipid containing atherosclerotic lesions. Since it has been recognized for many years that vascular injury in combination with hyperlipemia will result in rapid formation of atherosclerotic lesions, we attempted to combine vascular injury with hyperlipemia as a means of accelerating this process in the dog. Injury was produced in pulmonary arteries with experimental Dirofilaria immitis (DI or heartworm) infection. This filarial parasite produces characteristic lipid-free lesions containing smooth muscle cells and occasional monocytes and collagen. Plasma cholesterol was increased by feeding 10 dogs an essential fatty acid-deficient diet (EFAD) for 90 days. Five of the EFAD dogs were infected with 30 to 31 adult DI worms to produce pulmonary artery injury. The remaining 5 EFAD dogs were not subjected to any form of vascular injury. An additional 5 control dogs were not subjected to vascular injury nor to the EFAD diet. The arteries of dogs infected with DI developed myointimal proliferative lesions which contained smooth muscle cells and macrophages. In addition, the EFAD diet produced significant elevations in LDL but not VLDL plasma cholesterol in all 10 dogs fed the diet. However, the plasma cholesterol was less than 750 mg/dl in all EFAD-fed dogs. Although smooth muscle cells and macrophages in the pulmonary arteries of DI-infected dogs were focal points for lipid accumulation, cholesterol content of these injured arteries was not increased compared to noninjured EFAD dogs. The results suggest that even severe vascular injury does not reduce the threshold of 750 mg/dl required to produce significant lipid accumulation in canine arteries.