Hypercholesterolemic Pigs Research Articles

Cardiovascular dysfunction in hypercholesterolemia associated with enhanced formation of AT1-receptor and of eicosanoids.

In hypercholesterolemia with or without atherosclerosis cardiovascular dysfunction and altered signalling of angiotensin (Ang II), nitric oxide (NO), or prostanoids are intimately related to enhanced oxidant stress and concomitant changes in gene expression. We analyzed cardiac angiotensin receptor (AT1) expression and metabolism of Ang II, eicosanoids, and NO in hypercholesterolemic animals. Guinea pigs were fed a 1% cholesterol diet for 8 weeks (Chol). Hemodynamics were analyzed in Langendorff hearts. Spectrophotometric determination of plasma lipids and radioimmunological detection of eicosanoids/cyclic guanosine monophosphate (cGMP). Activities of NO synthase III (NOS-III) or angiotensin converting enzyme (ACE) were determined by enzymatic assays. AT1 receptor density was assessed by radioligand binding assay. NOS-III mRNAs were quantitated by reverse transcription polymerase chain reaction. Hypercholesterolemia was associated with fatty degeneration of the liver and profound myocardial and coronary (e.g., endothelial) dysfunction. In Chol Langendorff hearts we observed significant increases in coronary flow (26.0 +/- 1.0 vs. 17.5 +/- 0.5 mL/min/g tissue) but diminished coronary responses to bradykinin (Bk, 250 ng bolus) or adenosine (Ado, 250 micrograms bolus) (delta CPPBk/Ado: 5 +/- 0.5 vs. 7.2 +/- 1/0.9 +/- 0.1 vs. 1.9 +/- 0.3 cm2 (area under the curve)). AT1 receptor expression was significantly increased in Chol hearts (72 +/- 6.8 vs. 45 +/- 5.6 fmol/mg protein), whereas marked suppression of cardiac activities of ACE (1.96 +/- 0.34 vs. 4.90 +/- 0.20 nmol/min/mg tissue) and of the entire cardiac nitric oxide-cGMP axis (e.g., NOS-III activity: 1.9 +/- 0.4 vs. 3.1 +/- 0.1 pmol/min/mg tissue; NOS-III mRNA: 0.82 +/- 0.16 vs. 1.20 +/- 0.12 arbitrary units; cGMP release: 0.41 +/- 0.02 vs. 0.54 +/- 0.04 pmol/min/g tissue) were shown in Chol. Finally, cardiac release of eicosanoids prostacyclin (PGI2) and thromboxane (TxA2) were significantly enhanced (0.48 +/- 0.05 vs. 0.38 +/- 0.05 and 0.60 +/- 0.10 vs. 0.24 +/- 0.10 ng/min/g tissue, respectively). Enhanced cardiac PGI2 release and suppression of cGMP synthesis in Chol were even more pronounced on stimulation with Bk (38.2 +/- 3.0 vs. 28.2 +/- 2.0 ng/min/g tissue and 1.9 +/- 0.3 vs. 3.0 +/- 0.3 pmol/min/g tissue, respectively). Altered angiotensin-mediated signal transduction probably related to augmented eicosanoid formation does not compensate for the limited endogenous NO production and for cardiovascular dysfunction in hypercholesterolemic guinea pigs. In this context, changes in redox-sensitive regulation of gene expression (AT1 receptor, NOS-III--caused by enhanced oxidant stress--could play a pivotal role.

Blockade of angiotensin signaling improves myocardial function in hypercholesterolemia independent of changes in eicosanoid release.

In hypercholesterolemia in the presence or absence of atherosclerosis, cardiovascular dysfunction and altered signaling of angiotensin, nitric oxide, or prostanoids are closely related to enhanced oxidant stress. We analyzed the potentially beneficial effects of the specific angiotensin-converting enzyme inhibitor enalapril and the specific angiotensin receptor blocker losartan on cardiac performance, eicosanoid metabolism, and parameters of oxidant stress in hypercholesterolemic animals. Guinea pigs were fed a 1% cholesterol diet for 8 weeks (Chol) with or without equieffective doses of either enalapril (1.5 mg/kg/d; Ena) or losartan (3 mg/kg/d; Los). Hemodynamics were analyzed in Langendorff hearts. Detection of eicosanoids was by enzyme immunoassay. Estimation of plasma xanthine oxidase (XO) activity was determined by spectrophotometry. In hypercholesterolemic guinea pigs, enhanced oxidant stress (e.g., increased plasma XO activities) was associated with profound myocardial and coronary (e.g., endothelial) dysfunction. Both enalapril and losartan lowered plasma cholesterol levels slightly, but only the angiotensin receptor antagonist effectively suppressed the increased plasma XO activities (from 11.4 +/- 0.7 to 7.6 +/- 2.2 U/L), and at the same time decreased the augmented coronary flow (from 26.0 +/- 1.0 to 23.0 +/- 1.0 mL/min/g tissue) observed in hypercholesterolemic animals. Assessment of left ventricular pressure and contractility (e.g., dp/dtmax) as well as the diastolic relaxation parameter (tau) revealed substantial myocardial dysfunction (systolic and diastolic) in Chol that was more substantially (and comparably) improved during administration of losartan (Los) than during enalapril (Ena). Surprisingly, angiotensin signaling blockade by either antagonist further suppressed the diminished coronary dilator responses to bradykinin (BK; not significant for enalapril) or adenosine (Ado) was demonstrated in Chol Langendorff hearts [delta CPPBK/Ado: from 5.0 +/- 0.5/0.9 +/- 0.1 to 4.4 +/- 1.5/0.4 +/- 0.1 (Ena) or to 1.9 +/- 0.5/0.4 +/- 0.1 (Los) cm2 (area under the curve), respectively]. Finally, as expected from control studies using heart preparations from normocholesterolemic guinea pigs, enhanced cardiac release of eicosanoids, prostacyclin, and thromboxane in Chol (0.48 +/- 0.03 and 0.6 +/- 0.1 ng/min/g) was augmented even further by treatment with enalapril (Ena: 1.6 +/- 0.4 and 1.0 +/- 0.1 ng/min/g), but was significantly reduced to or below control levels in losartan-treated animals (Los: 0.4 +/- 0.1 and 0.2 +/- 0.1 ng/min/g). Blockade of angiotensin signaling via angiotensin-converting enzyme inhibition or receptor antagonism--although differentially acting on enhanced cardiac prostanoid metabolism and oxidant stress--efficiently restored proper systolic and diastolic myocardial performance (losartan was more beneficial than enalapril), probably by counterbalancing altered angiotensin II-->angiotensin receptor signaling in the cardiovascular system of hypercholesterolemic animals. Impaired coronary vasodilator capacity seems to be irreversible after 8 weeks of a high-cholesterol diet, as shown by the unexpected lack of a dilator effect with both enalapril and losartan.

Breakdown of plant carbohydrates in the digestive tract of pigs fed on wheat- or oat-based rolls

Cell wall materials from various cellular tissues of cereals may potentially influence the digestion and absorption processes in different ways. The objective of the present investigation was to study the breakdown of plant carbohydrates in the gastrointestinal tract of pigs fed a low-fibre (LF; 65 g kg−1 dry matter) wheat flour-based diet and two high-fibre diets with added insoluble fibre from wheat bran (HFWB; 112 g kg−1 dry matter) or soluble fibre from oat bran (HFOB; 108 g kg−1 dry matter). The diets were formulated to provide ∼0.31 energy from fat, ∼0.18 energy from protein and ∼0.51 energy from carbohydrates and were offered as baked rolls to eight ileum-cannulated hypercholesterolemic pigs in a crossover design. Carbohydrates were the predominant ileal constituent, with most of the carbohydrates present as non-starch polysaccharides (NSP). Starch in all diets was almost completely digested at this site of the gastrointestinal tract, with digestibility values above 0.99. The ingested amount of NSP was quantitatively recovered in ileal materials with the wheat-based diets, while there was a significant loss of 0.27 of NSP primarily as mixed linked (1→3)(1→4)--D-glucan with diet HFOB. The concentration and flow of lactic acid were also highest in the ileum after feeding diet HFOB, indicating that -glucan stimulated lactic acid formation. Any soluble components that reached the large intestine were almost completely broken down, while the digestibility of insoluble NSP constituents was significantly lower for diets LF and HFWB than for diet HFOB. The high degradation of oat bran resulted in a lower effect on faecal dry and wet bulking than was found with wheat bran. In conclusion, cereal starch was practically completely degraded in the small intestine, while the site and extent of NSP degradation, lactic acid formation and bulking properties were influenced by the fibre source. © 2000 Society of Chemical Industry

Identification of a novel Arg→Cys mutation in the LDL receptor that contributes to spontaneous hypercholesterolemia in pigs

We previously carried out genetic and metabolic studies in a partially inbred herd of pigs carrying cholesterol-elevating mutations. Quantitative pedigree analysis indicated that apolipoprotein (apo)B and a second major gene were responsible for the hypercholesterolemia in these animals. In this study, we assessed LDL receptor function by three different methods: ligand blots of liver membranes using β-very low density lipoprotein (VLDL) as a ligand; low density lipoprotein (LDL)-dependent proliferation of T-lymphocytes; and direct binding of 125I-labeled LDL to cultured skin fibroblasts. All three methods demonstrated that LDL receptor ligands bound with decreased affinity to the LDL receptor in these animals. In skin fibroblasts from the hypercholesterolemic pigs, the Kd of binding was about 4-fold higher than in cells from normal pigs. The cDNA of the pig LDL receptor from normal and hypercholesterolemic pigs was isolated and sequenced. We identified a missense mutation that results in an Arg→Cys substitution at the position corresponding to Arg94 of the human LDL receptor. The mutation is in the third repeat of the ligand binding domain of the receptor. By single-stranded conformational polymorphism (SSCP) analysis, we studied the relationship between LDL receptor genotype and plasma cholesterol phenotype. In contrast to humans, the hypercholesterolemia associated with the LDL receptor mutation in pigs was expressed as a recessive trait. The LDL receptor mutation made a far more significant contribution to hypercholesterolemia than did the apoB mutation, consistent with observations made in human subjects with apoB mutations. Within each genotypic group (mutated apoB or mutated receptor), there was a wide range in plasma cholesterol. As the animals were on a well-controlled low-fat diet, this suggests that there are additional genetic factors that influence the penetrance of cholesterol-elevating mutations. —Grunwald, K. A. A., K. Schueler, P. J. Uelmen, B. A. Lipton, M. Kaiser, K. Buhman, and A. D. Attie. Identification of a novel Arg→Cys mutation in the LDL receptor that contributes to spontaneous hypercholesterolemia in pigs. J. Lipid Res. 1999. 40: 475–485.

LDL Hypercholesterolemia Is Associated With Accumulation of Oxidized LDL, Atherosclerotic Plaque Growth, and Compensatory Vessel Enlargement in Coronary Arteries of Miniature Pigs

The association between accumulation of oxidized low density lipoprotein (LDL) and (1) progression of atherosclerotic plaques and (2) compensatory enlargement was assessed in the coronary arteries of LDL-hypercholesterolemic miniature pigs. In miniature pigs fed a 4% cholesterol diet, LDL cholesterol levels increased from 27+/-3.5 mg/dL (mean+/-SEM, n=36) to 250+/-28 mg/dL (n=10), 260+/-15 mg/dL (n=6), and 260+/-17 mg/dL (n=10) at 6, 14, and 24 weeks, respectively. Mean intimal areas of lesions in the left anterior descending coronary artery of hypercholesterolemic pigs were 0.16+/-0.046 mm2 at 6 weeks (n=10) and increased 5.4-fold (n=6, P<.05) and 10.6-fold (n=10, P<.001) at 14 and 24 weeks, respectively. Plaque growth was associated with an increase in mean internal elastic lamina area, from 1.44+/-0.17 to 4.38+/-0.52 mm2 (P=.007) and in mean luminal area from 1.42+/-0.15 mm2 in control pigs to 4.38+/-0.52 mm2 in pigs fed a cholesterol diet for 24 weeks (P=.007 vs control). Levels of total LDL in the intima, measured immunocytochemically, were 0.031+/-0.0098, 0.11+/-0.057 (P< or =.05), and 0.43+/-0.082 U (P<.001) at 6, 14, and 24 weeks, respectively. Corresponding levels of oxidized LDL were 0.034+/-0.023, 0.11+/-0.050 (P<.05), and 0.44+/-0.065 U (P<.001), respectively, suggesting that virtually all LDL in the intima is oxidized. Levels of oxidized LDL in the lesions were correlated with the intimal areas (r=.85, P<.0001) but were independent of plasma levels of LDL cholesterol and of oxidized LDL. Plaque levels of oxidized LDL were also correlated with internal elastic lamina areas (r=.72, P<.0001) and with luminal areas (r=.50, P=.0098). Plaque growth in the coronary arteries of LDL-hypercholesterolemic miniature pigs is associated with (1) an increase in plaque levels of oxidized LDL at constant plasma levels of LDL cholesterol and of oxidized LDL and (2) compensatory vessel enlargement proportional to plaque levels of oxidized LDL.

Enhanced endothelin-mediated coronary vasoconstriction and attenuated basal nitric oxide activity in experimental hypercholesterolemia.

Experimental hypercholesterolemia is associated with coronary vasomotor dysfunction. This study was designed to test the hypothesis that experimental hypercholesterolemia is characterized by altered coronary vasomotor responses to endothelin and inhibition of the endogenous NO pathway. Endothelin-1 (ET-1) at 5 ng x kg(-1) x min(-1) or N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of nitric oxide synthase (NOS), at 50 microg x kg(-1) x min(-1) was infused into the left anterior descending coronary artery in pigs before and after 10 weeks of cholesterol diet. There was a significant increase in serum cholesterol. At 10 weeks, ET-1 resulted in an accentuated decrease in coronary blood flow (CBF) and coronary artery diameter (CAD) compared with baseline (-88+/-6% versus -45+/-9%, P<.05, and -77+/-14% versus -18+/-8%, P<.05, respectively) and an increase in coronary vascular resistance (CVR) (242+/-18% versus 110+/-17%, P<.05); ET receptor density and binding affinity in epicardial coronary arteries were unchanged. The effect of L-NMMA on CBF, CAD, and CVR was attenuated at 10 weeks (-7+/-8% versus -48+/-4%, -2+/-3% versus -17+/-5%, and 16+/-10% versus 125+/-32%; each P<.05). Immunohistochemistry staining for constitutive NOS revealed a decrease in immunoreactivity in the coronary arteries of hypercholesterolemic pigs. The present study demonstrates an enhanced coronary vasoconstrictive response to pathophysiological doses of endothelin and an attenuated response to the inhibition of endogenous NO activity, suggesting an alteration in coronary vascular reactivity in experimental hypercholesterolemia.

The effect of basic fibroblast growth factor on coronary vascular tone in experimental hypercholesterolemia in vivo and in vitro.

To determine whether bFGF causes coronary vasoconstriction in experimental hypercholesterolemia in association with endothelial dysfunction. We infused 0.02 microgram/kg per min bFGF into the left anterior descending artery of anesthetized pigs (n = 5) before and after 10 weeks of a high-cholesterol diet. The coronary artery diameter (CAD) was measured by quantitative angiography and the coronary blood flow (CBF) was calculated using coronary-flow Doppler measurements. In additional in-vitro studies, epicardial arteries from normal and hypercholesterolemic pigs were exposed to cumulative concentrations of bradykinin (10(-11)-10(-6) mol/l) after precontraction with 10(-6) mol/l endothelin-1. Cumulative concentrations of bFGF (10(-13)-10(-8) mol/l) were added to other vessels with and without prior contraction with 10(-5) mol/l PGF2 alpha. Similar in-vitro studies were performed using rings from which the endothelium had been removed. In vivo, intracoronary administration of bFGF increased the CBF by 33 +/- 33% and the CAD by 12 +/- 5% at baseline, but reduced the CBF by 45 +/- 8% and the CAD by 14 +/- 6% after 10 weeks of a high-cholesterol diet. In vitro, the endothelium-dependent vasorelaxation in response to bradykinin was attenuated significantly in coronary arteries from hypercholesterolemic pigs (P < 0.05). Cumulative concentrations of bFGF exerted no effect on the coronary epicardial tone both in normal (with and without endothelium) and in hypercholesterolemic pigs. Acute administration of intracoronary bFGF caused coronary vasodilatation under steady-state conditions and vasoconstriction under diet-induced hypercholesterolemia. In vitro, bFGF did not affect the coronary epicardial tone. These findings suggest that the coronary vasoactive properties of bFGF may be exerted indirectly and are affected by the functional integrity of the endothelium.

Ascorbic Acid in Relation to Lipid Peroxidation in Hypercholesterolemic Guinea Pigs.

The effect of ascorbic acid on serum lipids and lipid peroxidation has been studied in guinea pigs kept on a high cholesterol diet for 6 and 16 weeks. After 6 weeks, total cholesterol (TC), low-density lipoprotein (LDL), very-low-density lipoprotein (VLDL), and triglycerides (TG) decreased significantly (p<0.001) in ascorbic acid-treated (groups 3 and 4) guinea pigs in comparison to their levels in hypercholesterolemic guinea pigs (group 2). Similar results for TC, LDL, and VLDL were observed after 16 weeks. However, the TG levels showed a nonsignificant decrease (NS) in group 3 and a significant decrease in group 4 (p<0.05) in comparison to those in group 2, after the 16 weeks study. The high-density lipoprotein (HDL) level after both 6 and 16 weeks showed a significant increase in the ascorbic acid-treated animals. The serum lipid peroxide level of animals fed both cholesterol and ascorbic acid showed a significant decrease (p<0.01). These results suggest that ascorbic acid lowers the serum level of cholesterol and protects lipids against peroxidative damage induced by hypercholesterolemia.

The Biologic Behavior of Balloon Hyperinflation–Induced Arterial Lesions in Hypercholesterolemic Pigs Depends on the Presence of Foam Cells

Lack of a large-animal model of accelerated atherosclerosis has limited study of the biologic behavior of atherosclerotic lesions. We hypothesized that mechanical vascular trauma combined with diet-induced hypercholesterolemia would result in rapid development of complex atherosclerosis-like lesions. Accordingly, we induced deep injury to a carotid artery by repetitive balloon hyperinflations in minipigs that were fed either an atherogenic diet (n = 30) or a standard diet (controls, n = 4) and examined the resultant lesions 1 month later. The neointimal lesions that evolved in 23 patent vessels from cholesterol-fed animals were complex, exhibiting infiltration of smooth muscle and foam cells and evidence of organized thrombus, recent thrombus, hemorrhage, and calcification. Lesions were separable histologically into two groups: foam-cell rich (n = 12), with 33 +/- 10 foam cells per high-power field, and foam-cell poor (n = 11), with 4 +/- 1 foam cells per high-power field. Minipigs with foam cell-rich lesions had higher serum cholesterol levels than those with foam cell-poor lesions (712 +/- 178 vs 468 +/- 240 mg/dL, P < .02). The incidence of intralesional thrombus was also significantly greater in foam cell-rich than in foam cell-poor lesions (50% vs 9%, P < .04). In addition, the degree of luminal stenosis was greater in the presence of lesions containing thrombus compared with those without thrombus (60 +/- 38% vs 30 +/- 29%, P = .05). Lesions in the control animals were fibrocellular and lacked foam cells and thrombus. Thus, hypercholesterolemia appeared to affect lesion composition and behavior.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibitory Effects of Aspirin on Coronary Hyperreactivity to Autacoids After Arterial Balloon Injury in Miniature Pigs

We examined the effects of aspirin on coronary hyperreactivity to autacoids after arterial balloon injury in miniature pigs. Coronary vasoconstriction induced by histamine and serotonin were examined angiographically before, 1 h, 1 week, and 1 month after balloon injury in 29 hypercholesterolemic miniature pigs. The animals were divided into three groups: group A, no treatment (n = 16); group B, pretreated with aspirin 50 mg/day for 2 days before injury (n = 7); and group C, treated with aspirin 50 mg/day for 2 days before and 5 days after injury (7 days in all) (n = 6). In group A, coronary vasoconstriction induced by autacoids was significantly greater at the injured than at the noninjured site at all times examined (p < 0.01). Hyperconstriction induced by the autacoids 1 h after injury were significantly less in groups B and C than in group A (p < 0.01). Hyperconstriction induced by autacoids 1 week after injury were significantly less in group B than in group A (p < 0.01) and were significantly less in group C than in group A (p < 0.01) or group B (p < 0.05). Treatment with aspirin for 2 or 7 days had no effect on the constrictive responses at the injured site 1 month after injury or on those at the noninjured site at all times examined. These results suggest that platelet-vessel wall interaction may play an important role in coronary hyperconstrictive responses to autacoids 1 h and 1 week after injury.

Circulating and tissue endothelin immunoreactivity in hypercholesterolemic pigs.

Hypercholesterolemia is characterized by a coronary vasoconstrictive response to the endothelium-dependent vasodilator acetylcholine. This abnormality may be due to reduced synthesis of endothelium-derived relaxing factor and/or enhanced synthesis and release of an endothelium-derived contracting factor. Endothelin is an endothelium-derived vasoconstrictor and mitogenic peptide that is present in normal plasma, and its circulating concentrations are elevated in disease states that are characterized by abnormal endothelium-dependent relaxation to acetylcholine. The current studies were designed to test the hypotheses that experimental hypercholesterolemia results in elevation of plasma and tissue endothelin immunoreactivity and that the abnormal acetylcholine-evoked coronary vasoconstriction in the hypercholesterolemic animals is associated with further elevation of plasma endothelin. Plasma concentrations and molecular forms of endothelin immunoreactivity were determined following 2% cholesterol diet for 4 months in pigs and during intracoronary acetylcholine administration. Second, we assessed the presence of endothelin in the coronary vascular wall by using immunohistochemistry. Hypercholesterolemia elevated plasma endothelin concentration and enhanced coronary artery tissue endothelin immunoreactivity. The endothelium-dependent vasodilator acetylcholine further increases plasma endothelin in hypercholesterolemia in association with coronary vasoconstriction. The predominant molecular form of endothelin in hypercholesterolemia is the biological active endothelin-1. This study suggests a role for endothelin as an early participant and a marker for the endothelial dysfunction in hypercholesterolemia as well as a participant in the atherogenic process.

Nucleotide sequence encoding the carboxyl-terminal half of apolipoprotein B from spontaneously hypercholesterolemic pigs

Previous studies from this laboratory characterized the hypercholesterolemia of pigs with a mutant allele of apolipoprotein B (apoB), designated Lpb5. This apoB allele is associated with low density lipoprotein (LDL) particles deficient in binding to the LDL receptor. To identify potential causative mutations in Lpb5 DNA, 10.6 kb of genomic DNA, encoding the carboxyl-terminal 58% of apoB were sequenced from the Lpb5 allele and from an allele encoding phenotypically normal apoB. Comparison of the two DNA sequences revealed 33 polymorphisms, 13 of which resulted in amino acid polymorphisms. To determine whether any of the amino acids at the polymorphic positions in Lpb5-encoded apoB were unique to that isoform, those positions were sequenced in four other pig apoB alleles encoding phenotypically normal apoB. None of the amino acids were by themselves uniquely encoded by the Lpb5 allele. However, a unique haplotype consisting of Asp3164 in conjunction with Ala3447 distinguished the Lpb5-encoded apoB from all other allelic isoforms sequenced in this region. To gain insight into changes in the tertiary structure of the mutant apoB, 13C-NMR analysis of LDL reductively methylated with [13C]-formaldehyde was performed. LDL has lysine residues that titrate at pH 10.5 and others that titrate at pH 8.9. The latter residues are thought to include those involved in the interaction of LDL with the LDL receptor. LDL from Lpb5 pigs possessed a smaller proportion of lysine residues titrating at pH 8.9 than did LDL from non-Lpb5 pigs, suggesting that the Lpb5-encoded apoB is altered in a manner affecting the microenvironment of particular lysine residues.

Inhibitory effects of heparin, aspirin and ketanserin on coronary artery vasoconstriction after arterial balloon injury in hypercholesterolemic miniature pigs

Objectives. The present study aimed to clarify the effects of heparin, aspirin and ketanserin on coronary artery vasoconstriction after arterial balloon injury.Background. The mechanisms of coronary artery vasoconstriction after coronary angioplasty are not well understood.Methods. After being fed a cholesterol-rich diet for 1 month, 71 Göttingen miniature pigs were randomly allotted to five groups; 16 pigs with no pretreatment (group A); 21 pigs pretreated with heparin, 3,000 U (group B); 13 pigs pretreated with aspirin, 50 mg/day orally for 2 days (group C); 11 pigs pretreated with ketanserin, 1 mg/kg body weight (group D); 10 pigs pretreated with aspirin, 50 mg/day for 2 days, heparin, 6,000 U and ketanserin, 1 mg/kg (group E). After this pretreatment, the left anterior descending or the left circumflex coronary artery, or both, was denuded by a 2F balloon catheter.Results. The coronary vasoconstriction at the injured sites reached a peak level 6 mil after the arterial injury and subsided within 30 min. The coronary vasoconstriction at the injured site 6 min after arterial injury was 56 ± 5% in group A, which was significantly greater than that in group B (28 ± 6%, p < 0.01), group C (25 ± 5%, p < 0.01), group D (26 ± 7%, p < 0.01) or group E (24 ± 5%, p < 0.01), whereas there was no significant difference in the coronary vasoconstriction among the latter four groups.Conclusions. These results suggest that serotonin released from aggregating platelets plays a major part in the platelet-dependent coronary artery vasoconstriction after arterial injury.

Antioxidant enzymes in cholesterol-induced atherosclerosis

Degradation of extracellular matrix, particularly interstitial collagen, promotes plaque instability and contributes to restenosis after vascular injury. We have explored the effects of vitamins C and E on the collagen content and metalloproteinase-1 (MMP-1) expression after angioplasty in hypercholesterolemic pigs. Iliac angioplasty was performed on 18 minipigs divided into three diet groups: a normal-cholesterol (NC), a high-cholesterol (HC) and a high-cholesterol plus vitamins C+E (HCV). Four weeks later, after sacrifice, the vascular collagen content and MMP-1 protein expression, along with the plasma caseinolytic activity and lipid peroxidation, were measured. MMP-1 was also determined in arterial rings stimulated with native low-density lipoproteins (LDL) isolated from experimental groups. Cholesterol-rich diet augmented plasma lipid peroxidation (P<0.05), reduced the collagen content and increased vascular MMP-1 expression after injury (P<0.05). Enhanced caseinolytic activity (identified as MMP-1) was also observed in HC plasma samples and in supernatants from arterial rings incubated with HC-LDL. Vitamins C and E markedly increased neointimal collagen content (P<0.01), reduced the hypercholesterolemia-induced changes in vascular MMP-1 (P<0.05) and diminished plasma and ex vivo caseinolytic activity. Vitamins C and E may help stabilize atherosclerotic plaque after angioplasty and favor vascular remodeling by increasing collagen content and reducing vascular MMP-1 expression in porcine hypercholesterolemia.

Nucleotide sequence encoding the carboxyl-terminal half of apolipoprotein B from spontaneously hypercholesterolemic pigs.

Nucleotide sequence encoding the carboxyl-terminal half of apolipoprotein B from spontaneously hypercholesterolemic pigs.

Low density lipoprotein heterogeneity in spontaneously hypercholesterolemic pigs

We previously described a strain of spontaneously hypercholesterolemic pigs carrying an apo-B allele termed Lpb5. Lpb5 pigs are heterogeneous with respect to the severity of their hypercholesterolemia. We have termed Lpb5 pigs with severe hypercholesterolemia Lpb 5.1 pigs, and those with moderate hypercholesterolemia Lpb 5.2, Lpb 5.1 animals have a dramatic increase in buoyant LDL relative to dense LDL, with a buoyant-to-dense LDL ratio of 2.2. In contrast, Lpb 5.2 and control pigs have buoyant-to-dense LDL ratios of 0.7 and 0.5 respectively. This ratio appears to be a stable characteristic of the Lpb 5.1 phenotype because sexually mature boars have a dramatic decrease in total plasma cholesterol concentration with no decrease in their ratio of buoyant-to-dense LDL. We have previously demonstrated a fourteen-fold overproduction of buoyant LDL in the Lpb 5.1 pigs, with very little conversion of dense LDL to buoyant LDL. In the current work, very low density lipoprotein (VLDL) turnover experiments were conducted to determine whether VLDL conversion to buoyant LDL was increased in the Lpb 5.1 pigs. VLDL conversion to buoyant LDL could not account for the increased production of buoyant LDL in Lpb 5.1 pigs. Thus, we cannot account for the increased production of buoyant LDL in the Lpb 5.1 pigs from any measurable plasma lipoprotein source. We have therefore termed this production of buoyant LDL in the Lpb 5.1 pigs direct buoyant LDL production.

Decreased lecithin: Cholesterol acyltransferase activity in the plasma of hypercholesterolemic pigs

Lecithin:cholesterol acyltransferase (LCAT) activity levels were determined, as function of plasma total cholesterol (TC) in 13 normocholesterolemic (TC less than 85 mg/dL) and in 28 hypercholesterolemic (TC greater than 98 mg/dL) pigs. The normocholesterolemic group consisted of pigs that carried apo-B allelic genes other than Lpb5 and or Lpb8. The hypercholesterolemic group consisted of Lpb5/x and Lpb5/8 heterozygous and Lpb5/5 homozygous animals. The data reported in this study show that the LCAT activity in the plasma of hypercholesterolemic (HC) pigs (79 +/- 43 units) was significantly lower (p less than 0.0005) compared to the normocholesterolemic controls (175 +/- 45 units). Furthermore, LCAT activity was positively correlated with TC in the normocholesterolemic group (r = +0.54; p less than 0.05), whereas it was negatively correlated with TC in the hypercholesterolemic group (r = -0.73; p less than 0.001). Additional data obtained from incubation experiments suggest that the lower LCAT activity in hypercholesterolemic pigs may be due, at least in part, to inhibition of LCAT activity by components found in the lipoprotein-deficient fractions of the plasma of hypercholesterolemic pigs.

Dietary tocotrienols reduce concentrations of plasma cholesterol, apolipoprotein B, thromboxane B2, and platelet factor 4 in pigs with inherited hyperlipemias

Normolipemic and genetically hypercholesterolemic pigs of defined lipoprotein genotype were fed a standard diet supplemented with 50 micrograms/g tocotrienol-rich fraction (TRF) isolated from palm oil. Hypercholesterolemic pigs fed the TRF supplement showed a 44% decrease in total serum cholesterol, a 60% decrease in low-density-lipoprotein (LDL)-cholesterol, and significant decreases in levels of apolipoprotein B (26%), thromboxane-B2 (41%), and platelet factor 4 (PF4; 29%). The declines in thromboxane B2 and PF4 suggest that TRF has a marked protective effect on the endothelium and platelet aggregation. The effect of the lipid-lowering diet persisted only in the hypercholesterolemic swine after 8 wk feeding of the control diet. These results support observations from previous studies on lowering plasma cholesterol in animals by tocotrienols, which are naturally occurring compounds in grain and palm oils and may have some effect on lowering plasma cholesterol in humans.

Overproduction of a buoyant low density lipoprotein subspecies in spontaneously hypercholesterolemic mutant pigs.

We previously described the hypercholesterolemia of pigs with defined apolipoprotein B (apo B) alleles associated with reduced binding of low density lipoprotein (LDL) to its receptor in vitro and slow clearance from the circulation in vivo. The increased plasma LDL in the hypercholesterolemic pigs was confined to a buoyant LDL subspecies. Because of this qualitative change in the LDL subspecies profile, we studied the turnover of buoyant and dense LDL subspecies independently. Normal and mutant radioiodinated buoyant and dense LDLs were simultaneously injected into normal and mutant pigs, and the clearance rates, interconversion rates, and production rates were determined. The sevenfold increase in buoyant LDL levels in the mutant pigs was due to a fivefold increase in buoyant LDL production. Total mutant LDL production was increased approximately 25%, suggesting that part of the increase in buoyant LDL production is at the expense of dense LDL production. Conversion of dense LDL to buoyant LDL made a small contribution to the buoyant LDL increase. The turnover analysis showed that dense LDL, in both mutant and control pigs, is primarily derived from a source other than buoyant LDL. To test this more directly, [3H]leucine was intravenously injected, and the specific activity of the LDL subspecies was measured over 96 hours. There was a large discrepancy in the areas under the specific activity-versus-time curves, indicating that buoyant LDL cannot be the sole precursor of dense LDL and further supporting the conclusion that buoyant and dense LDL are, in part, metabolically independent particles. These results show that genetic variation in the apo B locus can affect the synthetic rate of LDL and the LDL subspecies distribution.

Intramural hemorrhage and endothelial changes in atherosclerotic coronary artery after repetitive episodes of spasm in x-ray-irradiated hypercholesterolemic pigs.

To assess whether coronary spasm affects the progression of atherosclerosis and results in evolution of myocardial infarction, the role of coronary spasm on the fine structure of conduit coronary arteries was studied morphologically. Göttingen miniature pigs were fed a semisynthetic diet containing 2% cholesterol and 1.1% sodium cholate. One month after being on this diet, the pigs were anesthetized and the endothelium of a branch of the left coronary artery was denuded using a balloon catheter. X-ray irradiation in a dose of 1,500 rad was given twice selectively to the area denuded, after 4 and 5 months of cholesterol feeding. Five months after endothelial denudation, transient (group A) and repetitive episodes (group B) of coronary spasm were provoked by single and periodic (five times every 5 minutes) intracoronary injections of serotonin (10 micrograms/kg/injection), respectively. The extent of spasm by serotonin at the previously denuded site was 84 +/- 4% (n = 4) and 90 +/- 5% (n = 6) narrowing in groups A and B (p = NS between groups), respectively. Forty minutes after the final administration of serotonin, the left coronary artery was relaxed by nitroglycerin, and the heart was isolated and perfuse-fixed under physiological pressure. Intramural hemorrhage was noted at the spastic site in six pigs of group B but not in group A. The average percent luminal narrowing, on cross sections at the spastic site in group B, was significantly greater than in group A (56 +/- 7% vs. 23 +/- 5%, p less than 0.01). Scanning electron micrographs revealed that the endothelial lining was intact at the nonspastic site in both groups. In addition to the appearance of intercellular bridges at the spastic site in both groups, squeezing of endothelial cells and adhesion of white blood cells were present at the spastic site exclusively in group B. These findings are consistent with the hypothesis that repetitive spasm may have an important role in the progression of atherosclerosis and/or myocardial infarction.