Transgenic rabbits as models for atherosclerosis research

Several characteristics of the rabbit make it an excellent model for the study of lipoprotein metabolism and atherosclerosis. New Zealand White (NZW) rabbits have low plasma total cholesterol concentrations, high cholesteryl ester transfer protein activity, low hepatic lipase (HL) activity, and lack an analogue of human apolipoprotein (apo) A-II, providing a unique system in which to assess the effects of human transgenes on plasma lipoproteins and atherosclerosis susceptibility. Additionally, rabbit models of human lipoprotein disorders, such as the Watanabe Heritable Hyperlipidemic (WHHL) and St. Thomas' Hospital strains, models of familial hypercholesterolemia and familial combined hyperlipidemia, respectively, allow for the assessment of candidate genes for potential use in the treatment of dyslipoproteinemic patients. To date, transgenes for human apo(a), apoA-I, apoB, apoE2, apoE3, HL, and lecithin:cholesterol acyltransferase (LCAT), as well as for rabbit apolipoprotein B mRNA-editing enzyme catalytic poly-peptide 1 (APOBEC-1), have been expressed in NZW rabbits, whereas only those for human apoA-I and LCAT have been introduced into the WHHL background. All of these transgenes have been shown to have significant effects on plasma lipoprotein concentrations. In both NZW and WHHL rabbits, human apoA-I expression was associated with a significant reduction in the extent of aortic atherosclerosis, which was similarly the case for LCAT in rabbits having at least one functional LDL receptor allele. Conversely, expression of apoE2 in NZW rabbits caused increased susceptibility to atherosclerosis. These studies provide new insights into the mechanisms responsible for the development of atherosclerosis, emphasizing the strength of the rabbit model in cardiovascular disease research.     

Effect of coconut oil on plasma apo A-I levels in WHHL and NZW rabbits

Age-matched Watanabe (WHHL) and New Zealand White (NZW) rabbits were fed a coconut oil-enriched diet (14%, w/w) for 2 weeks. Lipid and apolipoprotein (apo) A-I levels in plasma and lipoprotein fractions were monitored. Within 3 days after the start of the coconut oil diet, plasma apo A-I and high-density lipoprotein (HDL)-apo A-I levels increased 3-fold in the WHHL rabbits. A smaller but significant increase (63%) in apo A-I and HDL-apo A-I levels was also observed in the NZW rabbits. HDL cholesterol levels also increased from 16 +/- 3 mg/dl during a regular diet to 46 +/- 16 mg/dl (288%) during the coconut oil diet in the WHHL rabbits and from 37 +/- 7 mg/dl to 69 +/- 19 mg/dl (186%), respectively, in the NZW rabbits. Apo A-I and HDL cholesterol levels fell sharply to the original levels soon after switching back to a regular diet (within 3 days for WHHL rabbits and within 5 days for NZW rabbits). The fractional catabolic rate calculated from 125I-HDL kinetic studies indicated that the turnover rate for HDL was significantly slower in WHHL rabbits fed the coconut oil diet than the control diet (0.018 +/- 0.004 h-1 vs. 0.027 +/- 0.007 h-1, P less than 0.01). No changes were found in the NZW rabbits fed either diet. Trilaurin, the main component of the coconut oil (46.9%) supplemented diet (6.5%, w/w), was also used in this study. The effect of trilaurin on plasma apo A-I and HDL-cholesterol levels is discussed.     

The distribution of elements in the tissues of Watanabe Heritable Hyperlipidemic rabbits

A deficiency or an excess of some elements in the diet is reported to modify the concentration of cholesterol in plasma, and, conversely, a reduction of cholesterol in the diet decreases zinc in plasma. We have studied the distribution of elements Na, K, Ca, Mg, Fe, Cu, Zn, S, P, and Mn in the tissues, plasma, heart, aorta, lung, liver, spleen, kidney, thymus, and brain of New Zealand White rabbits (NZW) and of Watanabe Heritable Hyperlipidemic rabbits (WHHL). The WHHL rabbits had a massive hypercholesterolemia (7.45 +/- 1.2 g/L) induced by a lack of liver low density lipoprotein receptors. The concentrations of elements in the tissues of the control NZW rabbits were very similar to those found in the normal rat. In WHHL, compared to NZW, besides the very important increase of total phosphorus in plasma explained by the augmentation of phospholipids, there was an increase of plasma copper (+44%) and zinc (+36%). The other noticeable changes were an increase of iron in heart (+19%), sulfur, and zinc in liver (+15% and +18%). The other changes observed in WHHL rabbits were, besides the increase of ceruloplasmin, the increase of vit E (+468%) and MDA (+62%). In conclusion, despite a massive increase of lipids in plasma, there was no major disturbance of element distribution in WHHL rabbits.     

Cholesterol synthesis in vivo and in vitro in the WHHL rabbit, an animal with defective low density lipoprotein receptors

These studies were undertaken to measure rates of synthesis of digitonin-precipitable sterols in vivo and in vitro in control rabbits (New Zealand (NZ) control) and in homozygous Watanabe heritable hyperlipidemic rabbits (WHHL) that lack receptors for low density lipoproteins (LDL). The plasma cholesterol concentration in NZ control fetuses equaled 79 mg/dl, rose to 315 mg/dl 12 days after birth, and fell to 80 mg/dl in young adult animals. At these same ages, cholesterol concentrations in the WHHL animals equal 315, 625, and 715 mg/dl, respectively. The rate of whole animal sterol synthesis in vivo, expressed as the mumol of [3H]water incorporated into sterols per hr per kg of body weight, was lower in the WHHL animals than in the NZ controls both in the fetuses (108 vs 176) and in the adult animals (48 vs 66). In adult NZ controls the content of newly synthesized sterols (rate of sterol synthesis) per g of tissue was highest in the liver (538 nmol/g per hr), adrenal gland (438), small bowel (371), and ovary (225) while lower rates of synthesis were found in 15 other tissues. In the WHHL rabbits a higher content of [3H]sterols was found only in the adrenal gland (2,215) while synthesis was suppressed in the liver (310), colon, lung, and kidney, and was unchanged in the remaining organs. These findings were confirmed by measurements of rates of sterol synthesis in the same tissues in vitro. When whole organ weight was taken into consideration, the tissues that were the major contributors to whole body sterol synthesis in both types of rabbits were liver, small bowel, skin, and carcass. However, it was the lower rate of synthesis in the liver of the WHHL animals that alone accounted for the lower rate of whole animal sterol synthesis seen in these rabbits. These studies demonstrate that in WHHL animals that lack LDL receptors and that have very high levels of circulating LDL cholesterol, the rate of cholesterol synthesis in nearly all tissues is normal but in the liver is significantly suppressed. Only the adrenal gland manifested enhanced synthesis. Such findings suggest that in the WHHL rabbit where LDL receptor activity is reduced and plasma LDL levels rise, mechanisms other than receptor-mediated LDL uptake may act to deliver cholesterol to the cells of the various organs and to the liver.     

Dissociation between cholesterol secretion and plasma lipid transfer activity in rabbits

Human and rabbit plasma contains a lipid transfer protein that transfers cholesteryl esters and triglycerides among the plasma lipoproteins and may also have a role in the movement of lipids into and out of cells. Little is known about the regulation of the activity of the lipid transfer protein, but in the rabbit, hypercholesterolemia is associated with increased plasma lipid transfer activity (LTA). Perfused rabbit livers secrete LTA, and hepatic cholesterol secretion is increased in rabbits with diet-induced hypercholesterolemia. Thus, experiments were performed with rabbits to determine if LTA is regulated by a concerted hepatic secretion of lipoprotein protein cholesterol and LTA. Rabbits were fed chow or chow plus coconut oil (14% wt/wt), and plasma lipids, LTA, and the rate of secretion of cholesterol into plasma were determined. Coconut oil feeding increased plasma cholesterol by 68%, LTA by 42%, and hepatic cholesterol secretion by 69%. Mevinolin (75 mg/day), an inhibitor of cholesterol biosynthesis, lowered LTA and plasma cholesterol without affecting the rate of secretion of cholesterol into plasma. These studies provide further evidence that, in the rabbit, plasma cholesterol and LTA are closely related, and the association is not likely to be caused by a concerted hepatic secretion of cholesterol and LTA.     

Control of variance in experimental studies of hyperlipidemia using the WHHL rabbit

Between-animal variability has frustrated many experimental studies in outbred animal models of human disease. Variability that arises from genetic heterozygosity can be minimized by use of experimental designs that match littermates (polyzygotic twins) across control and treatment groups. Poor breeding vigor has prevented use of this experimental design in the WHHL rabbit model of hyperlipidemia and atherosclerosis. A comparison of reproduction in WHHL and normal rabbits demonstrated that litter size is limited by functional deficits at ovulation, implantation, and gestation in WHHL females. Superovulation of females reliably produced expanded litters of WHHL rabbits. Plasma lipids were measured in expanded litters of Japanese White WHHL (JW-WW) and English Half-lop WHHL (EHL-WW) rabbits. The variance of plasma cholesterol within sibships was two- to three-fold less than that between-litters. Intraclass correlation of total cholesterol within litters of EHL-WW was 0.72 and within litters of JW-WW was 0.67. These data provide evidence of genetic modulation of hypercholesterolemia in WHHL rabbits and demonstrate that experimental designs in which littermates are paired across groups can decrease the number of animals needed or increase the sensitivity of hypothesis tests by two- to threefold.     

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.     

Characterization of arachidonic acid metabolism in Watanabe heritable hyperlipidemic (WHHL) and New Zealand white (NZW) rabbit aortas

WHHL rabbits develop progressive atherosclerosis. There are no visible signs of the disease at 1 month, however, by 12 months, the formation of aortic plaques is extensive. This study characterized arachidonic acid (AA) metabolism in 1 and 12 month old WHHL and NZW rabbit aortas. Vessels incubated with 14C-AA and A23187 metabolized AA to a number of oxygenated products as identified by high pressure liquid chromatography. The major AA metabolites produced by WHHL and NZW aortas were 6-keto PGF1 alpha, PGE2, 12- and 15-hydroxyeicosatetraenoic acids (HETEs). The structures of the HETEs were confirmed by gas chromatography-mass spectrometry. Indomethacin blocked the synthesis of prostaglandins (PGs) but not HETEs whereas ETYA, NDGA or removal of the endothelium attenuated the production of both PGs and HETEs. Measurement of 6-keto PGF1 alpha, 12- and 15-HETE by specific radioimmunoassays indicated that as the rabbits aged and as atherosclerosis progressed, aortas lost the ability to synthesize 6-keto PGF1 alpha and 15-HETE. Prior to the development of atherosclerosis, 1 month old WHHL aortas produced 70% less 15-HETE than did NZW aortas. Atherosclerotic aortas from 12 month old WHHLs synthesized 60% less 6-keto PGF1 alpha during stimulation with AA or A23187 than did 12 month old NZW aortas. We conclude that the development and expression of atherosclerosis in WHHL rabbits impairs the ability of aortas to metabolize AA to both PGs and HETEs.     

Role of receptor-independent low density lipoprotein transport in the maintenance of tissue cholesterol balance in the normal and WHHL rabbit

These studies were undertaken to determine the role of receptor-independent low density lipoprotein (LDL) transport in cholesterol balance across individual tissues and the whole animal. Homologous LDL, which measures total LDL transport, and methylated heterologous LDL, which measures receptor-independent LDL uptake, were cleared from the plasma at very different rates in the NZ control rabbit (3,900 and 1,010 microliter/hr per kg, respectively) whereas in the WHHL rabbit both preparations were cleared at essentially the same rate (approximately 1,070 microliter/hr per kg). Receptor-independent LDL clearance was detected in all tissues of the NZ control rabbit and these varied from 32 (spleen) to less than 0.5 (skeletal muscle) microliter/hr per g. In contrast, receptor-dependent LDL uptake was found in only about half of these same organs. In the WHHL rabbit, the rates of receptor-independent LDL transport were the same as in the NZ control rabbit, but no receptor-dependent uptake was detected. Using these clearance values it was calculated that in the control rabbit nearly 70% of LDL-cholesterol was removed from the plasma by the liver and 89% of this was receptor-mediated. With loss of receptor activity, however, the burden of LDL degradation was shifted away from the liver so that approximately 70% of LDL-cholesterol uptake took place in the extra-hepatic tissues of the WHHL rabbit. Thus, in the normal animal, the primary function of receptor-dependent LDL transport is to promote the rapid uptake and disposal of plasma LDL by the liver. In the absence of such receptor activity, cholesterol balance across most individual organs and the whole animal remains essentially normal and is mediated by the receptor-independent process. Because of the much lower absolute clearance rates manifested by this transport mechanism, however, substantial and predictable elevations in the circulating plasma LDL-cholesterol levels are required to maintain this balance.     

Defects of the LDL receptor in WHHL transgenic rabbits lead to a marked accumulation of plasma lipoprotein[a

In this study, we created LDL receptor (LDLr) defective (WHHL) transgenic rabbits expressing human apo[a] to examine whether LDLr mediates the Lp[a] clearance from the plasma. By crossbreeding WHHL rabbits with human apo[a] transgenic rabbits, we obtained two groups of human apo[a] transgenic rabbits with defective LDLr functions: apo[a](1/0) WHHL heterozygous (LDLr(+/-) and apo[a](+/0) WHHL homozygous (LDLr(-/-) rabbits. The lipid and lipoprotein levels of human apo[a] WHHL rabbits were compared to those of human apo[a] transgenic rabbits with normal LDLr functions (LDLr(+/+). The apo[a] production rate was evaluated by analyzing apo[a] mRNA expression in the liver, the major site for apo[a] synthesis in transgenic rabbits. We found that pre-beta lipoproteins were markedly increased accompanied by a 2-fold increase in the plasma Lp[a] in apo[a](+/0)/LDLr(+/-) rabbits and a 4.2-fold increase in apo[a](+/0)/LDLr(-/-) rabbits compared with that in apo[a](+/0) rabbits with normal LDLr function. In apo[a](+/0)/LDLr(-/-) rabbits, there was a marked increase in plasma total cholesterol and triglycerides, as was found in their counterpart non-transgenic WHHL rabbits. Northern blot analysis revealed that hepatic apo[a] expression in WHHL transgenic rabbits was similar to that in LDLr(+/+) transgenic rabbits, suggesting the accumulation of plasma Lp[a] in WHHL transgenic rabbits was not due to increased apo[a] synthesis.In conclusion, absence of a functional LDLr leads to a marked accumulation of plasma Lp[a] in human apo[a] transgenic WHHL rabbits and LDLr may participate in the catabolism of Lp[a] in rabbits.     

A novel mechanism by which probucol lowers low density lipoprotein levels demonstrated in the LDL receptor-deficient rabbit

Treatment of low density lipoprotein (LDL) receptor-deficient rabbits (WHHL rabbits) with probucol (1% w/w in a chow diet) lowered their LDL-cholesterol levels by 36%, consonant with the reported effectiveness of the drug in patients deficient in the LDL receptor. Initial studies of LDL fractional catabolic rate (FCR) using 125I-labeled LDL prepared from the serum of untreated WHHL rabbits showed no difference between probucol-treated WHHL rabbits and untreated WHHL rabbits. When, however, 125I-labeled LDL was prepared from donor WHHL rabbits under treatment with probucol and injected back into them, the FCR was found to be increased by about 50% above that measured simultaneously using 131I-labeled LDL prepared from untreated WHHL donors. The labeled LDL from probucol-treated donors was also metabolized more rapidly than that from untreated donors when injected into untreated WHHL rabbits or into untreated wild-type New Zealand White rabbits. Finally, it was shown that rabbit skin fibroblasts in culture degraded labeled LDL prepared from probucol-treated WHHL rabbits more rapidly than that prepared from untreated WHHL donors. This was true both for normal rabbit fibroblasts and also for WHHL skin fibroblasts, although the absolute degradation rates in the latter were, of course, much lower for both forms of LDL. The data indicate that a major mechanism by which probucol lowers LDL levels relates not to changes in the cellular mechanisms for LDL uptake or to changes in LDL production but rather to intrinsic changes in the structure and metabolism of the plasma LDL of the probucol-treated animal.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endothelium Dependent and Independent Relaxation of Aortic Rings from Watanabe Heritable Hyperlipidemic Rabbits after Exposure to a Free Radical Generating System

The effects of the xanthine oxidase/hypoxanthine free radical generating system on endothelium dependent and independent relaxation were compared in aortic rings from New Zealand white rabbits and heterozygous Watanabe heritable hyperlipidemic (WHHL) rabbits with mild atherosclerosis. Studies were carried out in young (3 months) and mature (18 months) animals. Plasma cholesterol levels were significantly higher in both 3 and 18 month WHHL animals. Endothelium independent relaxation to SNP did not differ between groups. However, the attenuation of relaxation to carbachol after xanthine oxidase/hypoxanthine treatment tended to be less in WHHL. This reached significance at 18 but not 3 months. We propose that this could be due to increases in levels of endogenous scavenger enzymes in these WHHL rabbits.     

D-galactosamine induced hepatitis in the rabbit: effect on lecithin:cholesterol acyltransferase activity, plasma lipid transfer protein activity and high density lipoproteins

Hepatitis was induced in rabbits by a single intraperitoneal injection of D(+)-galactosamine-HCl (750 mg/kg body wt). Plasma lecithin:cholesterol acyltransferase activity fell to 5% and lipid transfer protein activity to 50% of control values 48 hr after injection. Discoid high density lipoprotein began to appear in plasma of treated rabbits 36 hr after injection, along with populations of high density lipoprotein (HDL) which were both smaller (radius 3.7 nm) and larger (radius 5.9 nm) than the original HDL population (radius 4.8 nm).     

Relationship of vascular 15-lipoxygenase induction to atherosclerotic plaque formation in rabbits

Aortas from atherosclerotic rabbits have increased levels of 15-lipoxygenase, but the relationship between induction of this enzyme and the atherosclerotic process has not been defined. We found that dietary administration of cortisone acetate significantly suppressed atherosclerotic plaque formation in both Watanabe Heritable Hyperlipidemic (WHHL) and cholesterol-fed WHHL/NZW heterozygous rabbits. There was, however, no corresponding decrease in the elevated 15-lipoxygenase activity. In addition, the elevated 15-lipoxygenase activity in atherosclerotic rabbit aortas was uniformly distributed throughout the aorta, and was not preferentially localized in the lesions. These results indicate that induction of the 15-lipoxygenase is not necessarily causally related to plaque development, and that plaques are not the major source of the increased enzyme activity. However, the results confirm that hypercholesterolemia is a necessary condition for both atherosclerosis and 15-lipoxygenase induction, suggesting that perhaps the 15-lipoxygenase may represent a protective response to the hyperlipidemic stress. This possibility is supported by the finding that the induced 15-lipoxygenase converts linoleic acid, which is the predominant essential fatty acid in aorta, to 13-hydroxyoctadecadienoic acid (13-HODE). This compound is a chemorepellant factor for platelets, inhibits platelet thromboxane synthesis, and stimulates prostacyclin synthesis by endothelial cells.     

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.     

Effects of hyperlipidemia on the nursing ability of WHHL rabbits

The homozygous Watanabe heritable hyperlipidemic (WHHL) rabbit, an animal model for human familial hypercholesterolemia, which has been maintained in a closed colony, has a reproductive ability which is remarkably lower than that of normal rabbits. The present study was undertaken to determine whether this low reproductive ability was associated with hyperlipidemia, since it is not associated with inbreeding depression. WHHL dams with over 600 mg/dl of serum cholesterol level showed a weaning rate of only about 20%, while dams with about 300 mg/dl of cholesterol showed a 64% weaning rate. Both conception and weaning rate seemed to decrease with a rise in serum triglyceride. The weaning age of homozygous offspring from the homozygous WHHL dams was significantly higher than homozygous offspring from heterozygous WHHL dame. The rate of increase in body weight of the offspring from WHHL dams was significantly lower than that of the offspring from heterozygous dams under 24 days of age. We concluded that the low reproductive ability, especially low nursing ability, was associated with hyperlipidemia due to the deficiency of low density lipoprotein receptors.     

Exposure to ambient particles accelerates monocyte release from bone marrow in atherosclerotic rabbits

Exposure to air pollution [particulate matter, particles <10 microm (PM(10))] causes a systemic inflammatory response that includes stimulation of the bone marrow (BM) and progression of atherosclerosis. Monocytes are known to play a key role in atherogenesis by migration into subendothelial lesions where they appear as foam cells. The present study was designed to quantify the BM monocyte response in Watanabe heritable hyperlipidemic (WHHL) rabbits after PM(10) exposure. WHHL rabbits were given twice weekly intrapharyngeal instillations of 5 mg of PM(10) for 4 wk to a total of 40 mg and compared with control WHHL or New Zealand White (NZW) rabbits. The thymidine analog 5'-bromo-2'-deoxyuridine was used to label dividing cells in the BM and a monoclonal antibody to identify monocytes in peripheral blood. The transit time of monocytes through the BM was faster in WHHL than in NZW rabbits (30.4 +/- 1.9 h vs. 35.2 +/- 0.9 h, WHHL vs. NZW; P < 0.05). PM(10) instillation exposure increased circulating band cell counts, caused rapid release of monocytes from the BM, and further shortened their transit time through the BM to 23.2 +/- 1.6 h (P < 0.05). The percentage of alveolar macrophages containing particles in the lung correlated with the BM transit time of monocytes (r(2) = 0.45, P <0.05). We conclude that atherosclerosis increases the release of monocytes from the BM, and PM(10) exposure accelerates this process in relation to the amount of particles phagocytosed by alveolar macrophages.     

Lipoprotein(a) enhances advanced atherosclerosis and vascular calcification in WHHL transgenic rabbits expressing human apolipoprotein(a)

High lipoprotein(a) (Lp(a)) levels are a major risk factor for the development of atherosclerosis. The risk of elevated Lp(a) concentration is increased significantly in patients who also have high levels of low density lipoprotein (LDL) cholesterol. To test the hypothesis that increased plasma levels of Lp(a) may enhance the development of atherosclerosis in the setting of hypercholesterolemia, we generated Watanabe heritable hyperlipidemic (WHHL) transgenic (Tg) rabbits expressing human apolipoprotein(a) (apo(a)). We report here that Tg WHHL rabbits developed more extensive advanced atherosclerotic lesions than did non-Tg WHHL rabbits. In particular, the advanced atherosclerotic lesions in Tg WHHL rabbits were frequently associated with calcification, which was barely evident in non-Tg WHHL rabbits. To investigate the molecular mechanism of Lp(a)-induced vascular calcification, we examined the effect of human Lp(a) on cultured rabbit aortic smooth muscle cells and found that smooth muscle cells treated with Lp(a) showed increased alkaline phosphatase activity and enhanced calcium accumulation. These results demonstrate for the first time that Lp(a) accelerates advanced atherosclerotic lesion formation and may play an important role in vascular calcification.     

Concentration and distribution of apolipoproteins A-I and E in normolipidemic, WHHL and diet-induced hyperlipidemic rabbit sera.

Two sandwich-type enzyme immunoassays have been developed to measure apolipoproteins A-I and E in rabbit serum. Specific goat antibodies were purified by affinity chromatography and used both for coating and for preparing antibody-peroxydase conjugates. The sensitivity of these assays is sufficient to allow studies of apo A-I and E distribution in lipoproteins fractionated by gel filtration from 50 microliters of serum. In WHHL rabbits, apo A-I is 5-fold lower (5.2 +/- 2.5 mg/dl) and apo E is 8-fold higher (9.9 +/- 3.5 mg/dl) than in normolipidemic rabbits (29 +/- 4.3 mg/dl and 1.3 +/- 0.5 mg/dl, respectively). In hyperlipidemic rabbits, fed 2 months on a 0.5% cholesterol diet, the apo A-I level was similar (32 +/- 12 mg/dl) to that of normolipidemic rabbits, but the apo E level is 12-fold higher (15.1 +/- 5.5 mg/dl). In addition, HDL particles were enriched with cholesterol and apo E. The bulk of apo E and cholesterol is located in large beta-VLDL in diet-induced hyperlipidemia, whereas they are mainly located in smaller size beta-VLDL in WHHL rabbits. In normolipidemic rabbits apo E occurs mainly in HDL, and cholesterol is distributed in the main three lipoprotein fractions VLDL, LDL and HDL. Interestingly, HDL of WHHL rabbit are deficient in apo A-I. These results are compatible with profound perturbations of lipoprotein composition and metabolism in atherogenic hyperlipidemia.