Early embryonic lethality caused by targeted disruption of the 3-hydroxy-3-methylglutaryl-CoA reductase gene

The endoplasmic reticulum (ER) enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, which converts HMG-CoA to mevalonate, catalyzes the ratelimiting step in cholesterol biosynthesis. Because this mevalonate pathway also produces several non-sterol isoprenoid compounds, the level of HMG-CoA reductase activity may coordinate many cellular processes and functions. We used gene targeting to knock out the mouse HMG-CoA reductase gene. The heterozygous mutant mice (Hmgcr+/-) appeared normal in their development and gross anatomy and were fertile. Although HMG-CoA reductase activities were reduced in Hmgcr+/- embryonic fibroblasts, the enzyme activities and cholesterol biosynthesis remained unaffected in the liver from Hmgcr+/- mice, suggesting that the haploid amount of Hmgcr gene is not rate-limiting in the hepatic cholesterol homeostasis. Consistently, plasma lipoprotein profiles were similar between Hmgcr+/- and Hmgcr+/+ mice. In contrast, the embryos homozygous for the Hmgcr mutant allele were recovered at the blastocyst stage, but not at E8.5, indicating that HMG-CoA reductase is crucial for early development of the mouse embryos. The lethal phenotype was not completely rescued by supplementing the dams with mevalonate. Although it has been postulated that a second, peroxisome-specific HMG-CoA reductase could substitute for the ER reductase in vitro, we speculate that the putative peroxisomal reductase gene, if existed, does not fully compensate for the lack of the ER enzyme at least in embryogenesis.     

Apolipoprotein E4 in macrophages enhances atherogenesis in a low density lipoprotein receptor-dependent manner

Apolipoprotein E (apoE) and the low density lipoprotein receptor (LDLr) are well recognized determinants of atherosclerosis. In addition to hepatocytes, where both are highly expressed and contribute to plasma lipoprotein clearance, they are expressed in vascular cells and macrophages. In this study, we examined the effects of human apoE isoforms and LDLr levels in atherogenic pathways in primary macrophages ex vivo and atherosclerosis development after bone marrow transfer in vivo using mice expressing human apoE isoforms and different levels of LDLr expression. Increases in LDLr expression significantly increased cholesterol delivery into macrophages in culture, and the effects were more prominent with lipoproteins containing apoE4 than those containing apoE3. Conversely, increased LDLr expression reduced cholesterol efflux in macrophages expressing apoE4 but not in macrophages expressing apoE3. Furthermore, apoE3 protected VLDL from oxidation in vitro more than did apoE4. In LDLr-deficient mice expressing the human apoE4 isoform, Apoe4/4 Ldlr-/-, the replacement of bone marrow cells with those expressing LDLr increased atherosclerotic lesions in a dose-dependent manner compared with mice transplanted with cells having no LDLr. In contrast, atherosclerosis in Apoe3/3 Ldlr-/- mice, expressing the human apoE3 isoform, did not differ by the levels of macrophage LDLr expression. Our results demonstrate that apoE4, but not apoE3, in macrophages enhances atherosclerotic plaque development in mice in an LDLr-dependent manner and suggests that this interaction may contribute to the association of apoE4 with an increased cardiovascular risk in humans.     

The influence of dietary vitamin E, fat, and methionine on blood cholesterol profile, homocysteine levels, and oxidizability of low density lipoprotein in the gerbil

A 90-day feeding study with gerbils was conducted to evaluate the influence of dietary vitamin E levels (25 mg/kg diet, 75 mg/kg, 300 mg/kg, and 900 mg/kg), two levels of dietary methionione (casein or casein+L-methionine (1% w/w)) and two sources of lipid (soybean oil [20%] or soybean oil [4%]+coconut oil [16%, 1:4 w/w]) upon serum lipids (total cholesterol, HDL-cholesterol, LDL-cholesterol). In addition, this study examined the effects of diet-induced hyperhomocysteinemia and supplemental dietary vitamin E on the oxidation of low density lipoproteins. Tissue vitamin E (heart, liver, and plasma) demonstrated a dose response (P≤0.001) following the supplementation with increasing dietary vitamin E (25, 75, 300, and 900 mg/kg). In addition, tissue vitamin E levels were found to be higher (P≤0.001) in those animals receiving a combination of coconut oil+soybean oil as compared to the group receiving soybean oil solely. Blood cholesterol profiles indicated an increase (P≤0.001) in total cholesterol and LDL cholesterol by the influence of saturated fat and supplemental methionine. Low-density lipoprotein cholesterol profile demonstrated a reduction (P≤0.001) at the higher dietary vitamin E levels (300 and 900 mg/kg) as compared to the 25 mg/kg and 75 mg/kg dietary vitamin E. Plasma protein carbonyls were not influenced by dietary vitamin E nor by supplemental methionine intake. In vitro oxidation of LDL showed that vitamin E delayed the lag time of the oxidation phase (P≤0.001) and reduced total diene production (P≤0.001). On the contrary, supplemental methionine decreased (P≤0.001) the delay time of the lag phase, whereas total diene production was increased (P≤0.001). Plasma lipid hydroperoxides were significantly reduced (P≤0.05) with supplemental dietary vitamin E, whereas supplemental L-methionine (1%) resulted in a significant (P≤0.05) increase in lipid plasma hydroperoxide formation. Plasma homocysteine was elevated (P≤0.001) with supplemental dietary L-methionine (1%) as well as the inclusion of dietary saturated fat. The present data showed that 1) a combination of dietary lipids (saturated and unsaturated fatty acids) as well as vitamin E and methionine supplementation altered blood cholesterol lipoprotein profiles; 2) in vitro oxidation parameters including LDL (lag time and diene production) and plasma hydroperoxide formations were affected by vitamin E and methionine supplementation; and 3) plasma homocysteine concentrations were influenced by supplemental methionine and the inclusion of dietary saturated fat.     

Relative roles of LDLr and LRP in the metabolism of chylomicron remnants in genetically manipulated mice

Remnant-like emulsions labeled with cholesteryl [(13)C]-oleate were prepared with lipid compositions similar to remnants derived from triacylglycerol-rich lipoproteins. When injected into the bloodstream of conscious mice, the remnant-like emulsions were metabolized in the liver leading to the appearance of (13)CO(2) in the breath. Previously, using this technique, we found that remnant metabolism was significantly impaired but not completely inhibited in mice lacking low density lipoprotein receptors (LDLr). We have now found in mice with non-functional low density lipoprotein receptor-related protein (LRP) that breath enrichment of (13)CO(2) was significantly decreased, indicating that the LRP also plays an important role in the metabolism of chylomicron remnants (CR). The enrichment of (13)CO(2) in the expired breath was negligible in mice lacking both LDLr and receptor-associated protein (-/-), essential for normal function of LRP. In mice pre-injected with gluthatione S-transferase-receptor-associated protein to block LRP binding, there was a marked inhibition of the appearance of (13)CO(2) in the expired breath of homozygous LDLr-deficient mice, supporting the role of LRP in vivo. Whether or not LDLr were present, in mouse and human fibroblast cells human apoE3 or E4 but not apoE2 were essential for binding of remnant-like emulsions, while lactoferrin and suramin completely inhibited binding. We conclude that in normal mice LDLr are important for the physiological metabolism of CR. When LDLr are absent the evidence supports a role for the LRP in the uptake of CR in liver cells and in fibroblasts, with binding characteristics for CR-associated apoE similar to LDLr.     

Effect of dietary fish oil, vitamin E, and probucol on renal injury in the rat

Dietary fish oil, vitamin E, and probucol have been considered in a variety of human and experimental models of kidney disease. Using subtotal nephrectomized cholesterol-fed rats as a model for progressive kidney disease, we examined the effect of 5% dietary fish oil, or a combination of 5% dietary fish oil with 500 IU vitamin E/kg diet or 1% probucol on renal injury. Three-month-old Sprague Dawley rats were fed a control diet (C group) or a cholesterol supplemented (2%) diet (Ch group) containing either fish oil (FO group) or fish oil plus vitamin E (FO+E group) or fish oil plus probucol (FO+P group). After 4 weeks of dietary treatment, the right kidney was electrocoagulated and the left kidney nephrectomized. After 8 weeks, 24-hour urine was collected before sacrifice. No effect of the dietary treatments was noted on serum creatinine, blood urea nitrogen, or proteinuria, except that proteinuria was highest in FO+P group. Rats receiving the cholesterol diets had higher serum low density lipoprotein (LDL) + very low density lipoprotein (VLDL) cholesterol (P < 0.05). In contrast, rats in the FO+P group had the lowest serum total cholesterol and LDL+VLDL cholesterol among all groups. The FO group had 26% lower kidney alpha-tocopherol concentrations than the C group. However, inclusion of vitamin E in the diet (FO+E group) increased the kidney alpha-tocopherol status to a level comparable to that in the C group, whereas inclusion of probucol in fish oil diet (FO+P group) did not improve the kidney alpha-tocopherol status. Rats fed the cholesterol diet had a 2.5-fold higher glomerular segmental sclerosis (GSS) score and 1.5-fold higher glomerular macrophage (GM) subpopulation than the C group. These effects of the cholesterol diet were ameliorated by a fish oil diet (FO group: GSS by 30%, GM by 24%). The inclusion of vitamin E in the fish oil diet (FO+E group) did not further improve the GSS score or GM subpopulation. However, inclusion of probucol in fish oil diet (FO+P group) lowered the GSS score by 73% and reduced GM subpopulation by 83% compared with the Ch group. These remarkable changes can be attributed to the powerful hypocholesterolemic activity of probucol. Our findings indicate that progression of glomerular sclerosis in the rat remnant kidney model of progressive kidney disease can be significantly modulated with fish oil treatment.     

A 50% reduction of excitability but not of intercellular coupling affects conduction velocity restitution and activation delay in the mouse heart

Introduction

Computer simulations suggest that intercellular coupling is more robust than membrane excitability with regard to changes in and safety of conduction. Clinical studies indicate that SCN5A (excitability) and/or Connexin43 (Cx43, intercellular coupling) expression in heart disease is reduced by approximately 50%. In this retrospective study we assessed the effect of reduced membrane excitability or intercellular coupling on conduction in mouse models of reduced excitability or intercellular coupling.

Methods and Results

Epicardial activation mapping of LV and RV was performed on Langendorff-perfused mouse hearts having the following: 1) Reduced excitability: Scn5a haploinsufficient mice; and 2) reduced intercellular coupling: Cx43^CreER(T)/fl mice, uninduced (50% Cx43) or induced (10% Cx43) with Tamoxifen. Wild type (WT) littermates were used as control. Conduction velocity (CV) restitution and activation delay were determined longitudinal and transversal to fiber direction during S₁S₁ pacing and S₁S₂ premature stimulation until the effective refractory period. In both animal models, CV restitution and activation delay in LV were not changed compared to WT. In contrast, CV restitution decreased and activation delay increased in RV during conduction longitudinal but not transverse to fiber direction in Scn5a heterozygous animals compared to WT. In contrast, a 50% reduction of intercellular coupling did not affect either CV restitution or activation delay. A decrease of 90% Cx43, however, resulted in decreased CV restitution and increased activation delay in RV, but not LV.

Conclusion

Reducing excitability but not intercellular coupling by 50% affects CV restitution and activation delay in RV, indicating a higher safety factor for intercellular coupling than excitability in RV.     

Vitamin E in the prevention of cardiovascular disease: the importance of proper patient selection

Vitamin E is a naturally occurring fat-soluble antioxidant which has been proposed as a treatment for both primary and secondary protection against cardiovascular (CV) events. Promising data from observational epidemiological studies associating higher vitamin E dietary intake with lower risk of CV events have not been validated in randomized controlled clinical trials assessing the effect of vitamin E on CV outcomes. While the pendulum of medical opinion has swung to suggest that high dose vitamin E supplements have no place in the treatment and prevention of CV disease, new data is emerging that allows identification of a specific target population for this treatment, namely patients with diabetes mellitus and the haptoglobin genotype 2-2. This review details the scientific basis and clinical evidence related to the effect of vitamin E on CV outcomes, and the importance of proper patient selection in gaining therapeutic benefit from this intervention.     

Molecular analysis of voltage dependence of heterotypic gap junctions formed by connexins 26 and 32.

Heterotypic gap junctions formed by pairing Xenopus oocytes expressing hemichannels formed of Cx32 with those expressing hemichannels formed of Cx26 displayed novel transjunctional voltage (Vj) dependence not predicted by the behavior of these connexins in homotypic configurations. Rectification of initial and steady-state currents was observed. Relative positivity and negativity on the Cx26 side of the junction resulted in increased and decreased initial conductance (gj0), respectively. Only relative positivity on the Cx26 decreased steady-state conductance (gj infinity). This behavior suggested that interactions between hemichannels influences gap junction gating. The role of the first extracellular loop (E1) in these interactions was examined by pairing Cx32 and Cx26 with a chimeric connexin in which Cx32 E1 was replaced with Cx26 E1 (Cx32*26E1). Both junctions rectified with gj0/Vj relations that were less steep than that observed for Cx32/Cx26. Decreases in gj infinity occurred for either polarity Vj in the Cx32/Cx32*26E1 junction. Mutation of two amino acids in Cx26 E1 increased the steepness of both the gj0/Vj and gj infinity/Vj relations. These data demonstrate that fast rectification can arise from mismatched E1 domains and that E1 may contribute to the voltage sensing mechanisms underlying both fast and slow Vj-dependent processes.     

Changes in the cholesterol content of high-density plasma lipoproteins in guinea pigs in the presence of a raised exogenous cholesterol intake and different degrees of vitamin C saturation

The cholesterol content of the high-density plasma lipoproteins (d over 1.1 g.cm-3) of guinea-pigs with experimental vitamin C deficiency, followed by realimentation with suboptimal (1 mg/animal per day) or optimal (10 mg/animal per day) doses of L-ascorbic acid for 6-9 weeks in the continued presence of an elevated alimentary cholesterol intake (0.5 g/kg diet), did not exceed 5% of the total plasma cholesterol concentration and did not alter significantly with changes in the degree of vitamin C saturation of the organism. The maximum total body tissue cholesterol concentrations were found in C-deficient guinea-pigs (plasma, adrenals) and in the group with partial vitamin C deficiency (liver, brain); the lowest values were found in the group whose organism was fully saturated with vitamin C. Under conditions of a raised cholesterol intake, ascorbic acid stimulated its elimination from the organism, but did not affect the proportion of plasma cholesterol bound in high-density lipoproteins.     

Connexin 26 (GJB2) mutations, causing KID Syndrome, are associated with cell death due to calcium gating deregulation

The autosomic dominant KID Syndrome (MIM 148210), due to mutations in GJB2 (connexin 26, Cx26), is an ectodermal dysplasia with erythematous scaly skin lesions, keratitis and severe bilateral sensorineural deafness. The Cx26 protein is a component of gap junction channels in epithelia, including the cochlea, which coordinates the exchange of molecules and ions. Here, we demonstrate that different Cx26 mutants (Cx26D50N and Cx26G11E) cause cell death in vitro by the alteration of intra-cellular calcium concentrations. These results help to explain the pathogenesis of both the hearing and skin phenotypes, since calcium is also a potent regulator of the epidermal differentiation process.     

Influence of low-density lipoprotein (LDL) receptor on lipid composition, inflammation and parasitism during Toxoplasma gondii infection

Intracellular replication of Toxoplasma gondii requires cholesterol uptake by host cell low-density lipoprotein receptor (LDLr), a critical element in atherosclerosis. We evaluated host parasitism, inflammatory responses and development of atherosclerosis in LDLr knockout (LDLr(-/-)) and their controls C57BL/6 mice infected with T. gondii. Our results show that T. gondii cysts were reduced in LDLr(-/-) mice when compared to C57BL/6 mice. However, in presence of hypercholesterolemic diet, parasite growth in LDLr(-/-) mice was similar to that seen in infected C57BL/6 mice. In presence of a hypercholesterolemic diet, T. gondii infection leads to a 60% reduction of serum triacylglycerol, total and atherogenic lipoprotein cholesterol. When aortic valve lesion was analyzed, infected mice showed a reduction of atherosclerotic lesion area as well as CD36 expression. MCP-1, SRA-I, SRA-II, ICAM-1 and VCAM-1 mRNA expression was kept similar between infected and control groups. Thus, despite the intense inflammatory process, the drastic reduction in serum lipids seems to limit the development of atherosclerosis in LDLr(-/-) mice infected with T. gondii. In conclusion, our results indicate that T. gondii employs host LDLr to acquire cholesterol and favor its growth. However, in the presence of hypercholesterolemia, T. gondii parasites are able to acquire cholesterol-rich lipoproteins through an alternative host receptor, and overcome LDLr deficiency, favoring host parasitism and impairing lipid loading of foam cells.     

The effect of dietary menhaden,olive, and coconut oil fed with three levels of vitamin E on plasma and liver lipids and plasma fatty acid composition in rats

The effect of dietary fats with varying degrees of unsaturation in the presence of different concentrations of vitamin E on tissue lipid levels was studied in rats. Rats were fed either menhaden oil, olive oil or coconut oil at 15% levels with either 0.1, 0.3 or 0.6 mg/g of vitamin E as alpha-tocopherol for four weeks. Rat serum and liver were analyzed for total cholesterol, HDL-cholesterol, triacylglycerol and phospholipids. In addition, fatty acid composition of serum lipids was also analyzed. Serum total cholesterol and triacylglycerol were significantly lower in rats fed menhaden oil than in those fed olive or coconut oil, while the HDL-cholesterol was significantly higher in serum of rats fed menhaden and olive oil than in those fed coconut oil. Levels of vitamin E in the diet had only a significant effect on serum cholesterol and liver phospholipids. The Pearson correlation coefficient showed a significant positive relationship between serum triacylglycerol and total cholesterol, and a negative correlation between triacylglycerol and HDL-cholesterol, and between total and HDL-cholesterol.In the liver, total cholesterol was significantly higher in rats fed coconut oil than in rats fed menhaden oil. Total liver phospholipids were lower in rats fed either coconut oil or olive oil compared to those fed menhaden oil, especially with higher levels of vitamin E intake. Higher levels of vitamin E in the diet appear to increase triacylglycerol and phospholipids in livers of rats fed menhaden oil. In the liver a significant negative correlation was observed between phospholipids and cholesterol. The type and degree of unsaturation (polyunsaturated fatty acids in menhaden oil, monounsaturated fatty acids in olive oil and saturated fatty acids in coconut oil) significantly affected plasma and tissue lipids.     

Aberrant hemichannel properties of Cx26 mutations causing skin disease and deafness

Mutations in the human GJB2 gene, which encodes connexin26 (Cx26), underlie various forms of hereditary deafness and skin disease. While it has proven difficult to discern the exact pathological mechanisms that cause these disorders, studies have shown that the loss or abnormal function of Cx26 protein has a profound effect on tissue homeostasis. Here, we used the Xenopus oocyte expression system to examine the functional characteristics of a Cx26 mutation (G45E) that results in keratitis-ichthyosis-deafness syndrome (KIDS) with a fatal outcome. Our data showed that oocytes were able to express both wild-type Cx26 and its G45E variant, each of which formed hemichannels and gap junction channels. However, Cx26-G45E hemichannels displayed significantly greater whole cell currents than wild-type Cx26, leading to cell lysis and death. This severe phenotype could be rescued in the presence of elevated Ca²⁺ levels in the extracellular milieu. Cx26-G45E could also form intercellular channels with a similar efficiency as wild-type Cx26, however, with increased voltage sensitive gating. We also compared Cx26-G45E with a previously described Cx26 mutant, A40V, which has an overlapping human phenotype. We found that both dominant Cx26 mutants elicited similar functional consequences and that cells coexpressing mutant and wild-type connexins predominantly displayed mutant-like behavior. These data suggest that mutant hemichannels may act on cellular homeostasis in a manner that can be detrimental to the tissues in which they are expressed. connexin     

Apple Cider Vinegar Modulates Serum Lipid Profile,Erythrocyte, Kidney,and Liver Membrane Oxidative Stress in Ovariectomized Mice Fed High Cholesterol

The purpose of this study was to investigate the potentially beneficial effects of apple cider vinegar (ACV) supplementation on serum triglycerides, total cholesterol, liver and kidney membrane lipid peroxidation, and antioxidant levels in ovariectomized (OVX) mice fed high cholesterol. Four groups of ten female mice were treated as follows: Group I received no treatment and was used as control. Group II was OVX mice. Group III received ACV intragastrically (0.6 % of feed), and group IV was OVX and was treated with ACV as described for group III. The treatment was continued for 28 days, during which the mice were fed a high-cholesterol diet. The lipid peroxidation levels in erythrocyte, liver and kidney, triglycerides, total, and VLDL cholesterol levels in serum were higher in the OVX group than in groups III and IV. The levels of vitamin E in liver, the kidney and erythrocyte glutathione peroxidase (GSH-Px), and erythrocyte-reduced glutathione (GSH) were decreased in group II. The GSH-Px, vitamin C, E, and β-carotene, and the erythrocyte GSH and GSH-Px values were higher in kidney of groups III and IV, but in liver the vitamin E and β-carotene concentrations were decreased. In conclusion, ACV induced a protective effect against erythrocyte, kidney, and liver oxidative injury, and lowered the serum lipid levels in mice fed high cholesterol, suggesting that it possesses oxidative stress scavenging effects, inhibits lipid peroxidation, and increases the levels of antioxidant enzymes and vitamin.     

Maternal intake of vitamin E and birth defects,national birth defects prevention study, 1997 to 2005

BACKGROUND In a recent study, high maternal periconceptional intake of vitamin E was found to be associated with risk of congenital heart defects (CHDs). To explore this association further, we investigated the association between total daily vitamin E intake and selected birth defects. METHODS: We analyzed data from 4525 controls and 8665 cases from the 1997 to 2005 National Birth Defects Prevention Study. We categorized estimated periconceptional energy‐adjusted total daily vitamin E intake from diet and supplements into quartiles (referent, lowest quartile). Associations between quartiles of energy‐adjusted vitamin E intake and selected birth defects were adjusted for demographic, lifestyle, and nutritional factors. RESULTS: We observed a statistically significant association with the third quartile of vitamin E intake (odds ratio [OR], 1.17; 95% confidence interval [CI], 1.01–1.35) and all CHDs combined. Among CHD sub‐types, we observed associations with left ventricular outflow tract obstruction defects, and its sub‐type, coarctation of the aorta and the third quartile of vitamin E intake. Among defects other than CHDs, we observed associations between anorectal atresia and the third quartile of vitamin E intake (OR, 1.66; 95% CI, 1.01–2.72) and hypospadias and the fourth quartile of vitamin E intake (OR, 1.42; 95% CI, 1.09–1.87). CONCLUSION: Selected quartiles of energy‐adjusted estimated total daily vitamin E intake were associated with selected birth defects. However, because these few associations did not exhibit exposure‐response patterns consistent with increasing risk associated with increasing intake of vitamin E, further studies are warranted to corroborate our findings. Birth Defects Research (Part A), 100:647–657, 2014. © 2014 Wiley Periodicals, Inc.     

Cx43 contributes to TGF-β signaling to regulate differentiation of cardiac fibroblasts into myofibroblasts

Differentiation and activation of fibroblasts into myofibroblasts which express α-smooth muscle actin (α-SMA) are essential for wound healing and tissue repair. Change in fibroblast properties is initiated by transforming growth factor β (TGF-β). Here, we sought to investigate whether connexin43 (Cx43), a gap-junctional protein, contributes to differentiation of cardiac fibroblasts to myofibroblasts. In cultured neonatal rat cardiac fibroblasts, we found that expression of α-SMA increases in parallel with Cx43 by using immunocytochemistry, and that knockdown of the endogenous Cx43 activity with antisense oligodeoxynucleotides (AS) inhibits α-SMA expression significantly, while overexpression of Cx43 increases α-SMA expression remarkably. These findings demonstrate that Cx43 contributes to TGF-β signaling to regulate α-SMA expression. Thus, we propose a novel physiologic function of Cx43, which plays a critical role in the pathological activation of cardiac fibroblasts in the myocardial fibrosis associated with heart failure.     

Calorie restriction attenuates LPS-induced sickness behavior and shifts hypothalamic signaling pathways to an anti-inflammatory bias

Calorie restriction (CR) has been demonstrated to alter cytokine levels; however, its potential to modify sickness behavior (fever, anorexia, cachexia) has not. The effect of CR on sickness behavior was examined in male C57BL/6J mice fed ad libitum or restricted 25% (CR25%) or restricted 50% (CR50%) in food intake for 28 days and injected with 50 μg/kg of LPS on day 29. Changes in body temperature, locomotor activity, body weight, and food intake were determined. A separate cohort of mice were fed ad libitum or CR50% for 28 days, and hypothalamic mRNA expression of inhibitory factor κB-α (IκB-α), cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)), suppressor of cytokine signaling 3 (SOCS3), IL-10, neuropeptide Y (NPY), leptin, proopiomelanocortin (POMC), and corticotrophin-releasing hormone (CRH) were determined at 0, 2, and 4 h post-LPS. CR50% mice did not develop fevers, whereas the CR25% mice displayed a fever shorter in duration but with the same peak as the controls. Both CR25% and CR50% mice showed no sign of anorexia and reduced cachexia after LPS administration. Hypothalamic mRNA expression of NPY and CRH were both increased by severalfold in CR50% animals preinjection compared with controls. The CR50% mice did not demonstrate the expected rise in hypothalamic mRNA expression of COX-2, microsomal prostaglandin E synthase-1, POMC, or CRH 2 h post-LPS, and leptin expression was decreased at this time point. Increases in SOCS3, IL-10, and IκB-α expression in CR50% animals were enhanced compared with ad libitum-fed controls at 4 h post-LPS. CR results in a suppression of sickness behavior in a dose-dependent manner, which may be due to CR attenuating proinflammatory pathways and enhancing anti-inflammatory pathways.     

Effects of the calcium channel blocker amlodipine on serum and aortic cholesterol,lipid peroxidation,antioxidant status and aortic histology in cholesterol-fed rabbits

Reactive oxygen metabolites and oxidized fatty acids are proinflammatory and are involved in the pathophysiology of atherosclerosis. Amlodipine, a unique third-generation dihydropyridine-type calcium channel blocker, seems to exert atheroprotective effects through its antioxidant properties related to its chemical structure and independent of its calcium channel-blocking effect. In this study, the interactions of amlodipine with major cellular antioxidants were investigated in order to elucidate the mechanisms underlying its atheroprotective effects. New Zealand white male rabbits were fed regular chow (group 1), chow with 1% cholesterol (group 2), regular chow plus 5 mg/kg/day amlodipine per os (group 3) and 1% cholesterol plus amlodipine (group 4) for 8 weeks. Total cholesterol, malondialdehyde (MDA) and vitamin E concentrations and catalase and superoxide dismutase (SOD) activities were determined in blood drawn before and after the experimental period. Aortic tissue was examined for atherosclerotic changes and aortic total cholesterol, MDA, catalase and SOD were determined. At the end of the 8-week treatment period, serum total cholesterol and plasma MDA were elevated in groups 2 and 4. In group 2, serum vitamin E and plasma SOD diminished (p < 0.05) and catalase increased (p < 0.05). In group 4, SOD activity increased at the end of treatment. MDA levels were lower and plasma SOD activities were higher in group 4 than in group 2. Aortic tissue investigations revealed higher total cholesterol and MDA concentrations and catalase activities in group 2 than in group 4, and the highest tissue SOD activity was recorded in group 4 (p < 0.05 for all comparisons). Morphological examination of aortic tissues exhibited endothelial disarrangement and lipid deposition in group 2. Histopathological alterations related to atherogenesis were less in group 4 than in group 2. Amlodipine seems to exert atheroprotective effects by reducing aortic cholesterol accumulation and blood and aortic lipid peroxidation, enhancing SOD activity both in blood and aortic tissue and suppressing the consumption of vitamin E. On the other hand, the suppression of catalase activity in blood and the aorta interferes with the drug's well-known antioxidant effects.