Effects of cholesterol diets on vascular function and atherogenesis in rabbits

Vascular endothelial dysfunction is an important early event in atherogenesis. To evaluate the effects of different levels of cholesterol-containing diets on vascular function and atherogenesis, 17 New Zealand White male rabbits were randomized into four groups: Control with noncholesterol, 10-week 0.5% (0.5C-10) or 1% cholesterol (1C-10), and 14-week 0.5% cholesterol (0.5C-14) feedings. After 10 or 14 weeks, the aortas were harvested for studies of vascular endothelial function and percentage surface lipid lesions. The 0.5% and 1% cholesterol feedings resulted in the same degree of hypercholesterolemia independent of the level and period of cholesterol feeding. There was a decreased trend in vascular endothelial-dependent relaxation to acetylcholine in cholesterol-fed rabbits. Fourteen-week cholesterol feeding induced the least vascular dilation at a concentration of 10-7 M acetylcholine (-38 +/- 3%, -23 +/- 4%, -23 +/- 2%, and -15 +/- 5% in control, 0.5C-10, 1C-10, and 0.5C-14 groups, respectively, P = 0.003). More cumulative exposure of arterial walls to cholesterol induced more surface lipid lesions in the aorta (r = 0.877, P < 0.001). There was a negative relationship between aortic lesions and vasodilation (r = -0.557, P = 0.020 for calcium ionophore; r = -0.463, P = 0.062 for acetylcholine). We conclude that the 0.5% and 1% cholesterol feedings induce similar degrees of hypercholesterolemia. However, aortic lipid lesions and vascular reactivity are dependent on cumulative exposure to cholesterol rather than serum cholesterol level only. Furthermore, decreased vascular endothelial relaxation in cholesterol-fed rabbits was related to lipid plaques in the aorta.     

Failure of L-carnitine to protect mice against ammonia toxicity

Recent reports indicate that intraperitoneal administration of L-carnitine protects mice from ammonia toxicity. We found that mice injected with L-carnitine and subsequently challenged with ammonium acetate succumb as readily as mice injected with saline and the ammonium acetate. Mice pretreated with L-carnitine exhibited higher levels of liver ammonia than the saline-pretreated control mice. The ammonia and urea levels in serum and brains were similar in two groups. Our findings are in contrast to those reported previously and therefore warrants further investigation before L-carnitine can be considered as a drug to alleviate hyperammonemia in humans.     

Failure of selenomethionine residues in albumin and immunoglobulin G to protect against peroxynitrite.

Selenomethionine has been suggested to protect against peroxynitrite by quenching it in vivo. Selenomethionine is distributed randomly in the methionine pool. Albumin and IgG were purified from plasma of a human being before and after 28 days of supplementation with 400 microg selenium/day as selenomethionine. The albumin contained 1 selenium atom, presumably as selenomethionine, per 8000 methionine residues before supplementation and 1 per 2800 after supplementation. Although this ratio suggested that selenomethionine would not have as great an effect in quenching peroxynitrite as would methionine, direct testing of the albumin and IgG fractions was carried out to assess the ability of these proteins to prevent peroxynitrite oxidation of dihydrorhodamine 123 to rhodamine 123. The ability of the albumin preparations to resist nitration of tyrosine residues was also assessed. The high-selenomethionine preparations of the proteins had no greater effect in quenching the peroxynitrite than did the normal-selenomethionine preparations. These results do not support the proposal that selenomethionine in proteins contributes to in vivo protection against peroxynitrite.     

Failure of N-acetylcysteine to protect against cis-dichlorodiammineplatinum(II)-induced hematopoietic toxicity in mice

In view of the results showing a decrease in cis-dichlorodiammineplatinum(II) (cis-DDP) nephrotoxicity after administration of thiol donors, this study was carried out to test the possibility that N-acetylcysteine (NAC) was active against myelodepressive effects of the anticancer drug. Cis-DDP (15.5 mg/kg body weight, i.v.) was administered to control mice and to mice treated simultaneously or 1 h later with NAC (800 mg/kg body weight, i.v.). At various times after treatment, up to 11 days, assessments were made of peripheral blood cell levels and bone marrow progenitor cell (CFUs and CFUc) concentrations. Cis-DDP caused a decrease in hemopoietic precursor cells in the order of that caused by other hemopoietic precursor cells in the order of that caused by other myelodepressive drugs, whereas there was only a slight decrease in peripheral blood WBC. In this experimental setting, NAC administration did not afford significant protection against platinum toxicity on bone marrow precursors or peripheral blood cells.     

Transforming growth factor-beta1 gene and protein expression associated with atherogenesis of cholesterol-fed rabbits

Transforming growth factor-beta1 (TGF-1beta) has been shown to modulate both cell proliferation and the synthesis of extracellular matrix by vascular cells. This study was aimed to establish the temporal correlation between TGF-beta1 expression, the expression of the extracellular matrix protein fibronectin, and plaque development during atherogenesis of hypercholesterolemic rabbits. New Zealand White rabbits were fed with 2% cholesterol-supplemented chow for 1 week, 2 weeks, 3 weeks or 6 weeks. TGF-beta1 mRNA and protein expression was examined in serial sections of aorta by in situ hybridization and immunohistochemistry. Fibronectin expression was examined by immunohistochemistry. In the control and 1-week feeding group, the expression of TGF-beta1 mRNA and protein was not apparent. In 2-week feeding group, intimal thickening was detected in which TGF-beta1 mRNA and protein were not clearly observed, either. The 3-week and 6-week feeding groups exhibited fatty streaks in which TGF-beta1 mRNA and protein expression markedly increased as feeding proceeded. Cell type-specific staining indicated that TGF-beta1 was expressed by macrophages as well as smooth muscle cells of the fatty streaks. Immunostaining of fibronectin detected low expression levels in control, 1-week and 2-week feeding groups with pronounced upregulation in the thickened intima and the proximal media in 3-week and 6-week feeding groups. These results implicate a role for TGF-beta1 in modulating fatty streak formation and the synthesis of extracellular protein fibronectin during plaque development.     

Identification of Fasciola hepatica recombinant 15-kDa fatty acid-binding protein T-cell epitopes that protect against experimental fascioliasis in rabbits and mice

Vaccination with fatty acid-binding proteins (FABPs) from Fasciola hepatica has been shown to confer significant levels of protection against challenge infection in mice, rabbits, and sheep. A recombinant 15-kDa FABP (rFh15) has been purified and also shown to be an immunoprotective molecule. From the rFh15 molecule sequence 2, 12- and 10-mer putative T-cell epitopes were identified, the first an Fh15Ta of amino acid sequence IKMVSSLKTKIT, and the second an Fh15Tb of amino acid sequence VKAVTTLLKA. The synthesized oligonucleotides were cloned individually into a pGEX-2TK expression vector. The overexpressed fusion protein was affinity purified using glutathione S-transferase (GST) by competitive elution with excess reduced glutathione. These GST fusion proteins were emulsified in Freund adjuvant for rabbit immunizations or further purified as peptides after digestion with thrombin. The purified 12- and 10-mer peptides were either emulsified in Freund adjuvant for immunizations in rabbits or used in an adjuvant-adaptation (ADAD) system, followed by challenge infection with F. hepatica metacercariae in mice and rabbits. In vaccinated-challenged rabbits, the highest levels of protection were found in those treated with GST-epitopes (Fh15Ta 48.2% and Fh15Tb 59.1% reduction, respectively), as compared to GST-immunized controls. Moreover, those immunized with Fh15Ta had higher (84%) numbers of immature flukes as compared with Fh15Tb (41%) or GST alone (64%). The rabbits immunized with the putative T-cell epitopes in adjuvant had a 13% reduction in flukes in those with Fh15Ta and also were highest with immature flukes (46%). In vaccinated mice challenged with a lethal number of metacercariae, both CD-1 and BALB/c mice treated with complete ADAD-GST-Ta had the highest (40%) survival rates of all groups by 47 days postinfection. Thus the Fh15Ta and Fh15Tb polypeptide epitopes warrant further study as a potential vaccine against F. hepatica. Antibody isotype studies in mice revealed a mixed Thl/Th2 response to vaccination.     

Cholesterol hydroperoxides inhibit calmodulin and suppress atherogenesis in rabbits

A mixture of cholesterol autoxidation products, prepared from an aged sample of cholesterol by recrystallization from methanol, inhibits calmodulin irreversibly in a Ca2+-dependent reaction. Inhibitory activity is lost after treatment with NaBH4, NaCNBH3, or NaI, from which we conclude that calmodulin inhibition is due to one or more cholesterol hydroperoxides. Partially purified cholesterol hydroperoxides, with or without cholesterol, were fed to young adult white rabbits. Cholesterol in the diet caused extensive atheroma formation in the aortas, but the addition of cholesterol hydroperoxides markedly reduced lesion formation. A cholesterol hydroperoxide preparation that was reduced by treatment with NaI was not effective in preventing atheroma formation. Cholesterol hydroperoxides did not lower cholesterol concentrations in blood plasma, liver, or heart.     

Lipid peroxidation--the factor promoting cholesterol accumulation in cells in atherogenesis

The cholesterol transfer between human erythrocytes and main classes of serum lipoproteins (LP) from healthy donors and artery-coronary disease patients was studied (artery-coronary disease is the main manifestation of atherosclerosis). It is shown that low-density lipoproteins (LDL) are capable of transporting cholesterol to erythrocytes, which lack the specific receptors for LDL. The cell cholesterol content in comparison with erythrocytes incubated without LDL was increased by 11.4%. The effect was even higher in case of LDL, isolated from serum of artery-coronary subjects (the cell cholesterol content was increased by 33.8%). High-density lipoproteins (HDL) accept cholesterol from cell membranes. However, cholesterol-accepting properties of HDL from artery-coronary disease patients were suppressed as compared with normal HDL. Both discovered events must promote the cholesterol accumulation in cell membranes in atherosclerosis. As it is shown by the spin probe method, lipid peroxidation (LPO) causes the disturbance of the structural organization of LP and as the consequence of that--the increase of LDL cholesterol-donating ability and the decrease of HDL cholesterol-accepting ability. The greater LDL are oxidized, the more cholesterol they transport to erythrocytes during incubation. The greater is the level of HDL peroxidation, the stronger their cholesterol-accepting function is suppressed. These results suggest that LPO can play an important role in LP modification, the disturbance of their interaction with cell surface and the cholesterol accumulation in cells in atherosclerosis.     

Human C-reactive protein does not protect against acute lipopolysaccharide challenge in mice

The physiological and pathophysiological functions of C-reactive protein (CRP), the classical acute-phase protein, are not well established, despite many reports of biological effects of CRP in vitro and in model systems in vivo. Limited, small scale experiments have suggested that rabbit and human CRP may both protect mice against lethal toxicity of Gram-negative bacterial LPS. However, in substantial well-controlled studies in C57BL/6 mice challenged with Escherichia coli O111:B4 LPS, we show in this work that significant protection against lethality was conferred neither by an autologous acute-phase response to sterile inflammatory stimuli given to wild-type mice 24 h before LPS challenge, nor by human CRP, whether passively administered or expressed transgenically. Male mice transgenic for human CRP, which mount a major acute-phase response of human CRP after LPS injection, were also not protected against the lethality of LPS from either E. coli O55:B5 or Salmonella typhimurium. Even when the acute-phase human CRP response was actively stimulated in transgenic mice before LPS challenge, no protection against LPS toxicity was observed. Indeed, male mice transgenic for human CRP that were pretreated with casein to stimulate an acute-phase response 24 h before LPS challenge suffered significantly greater mortality than unstimulated human CRP transgenic controls. Rather than being protective in this situation, human CRP may thus have pathogenic proinflammatory effects in vivo.