Lanthanum carbonate, like sevelamer-HCl, retards the progression of vascular calcification and atherosclerosis in uremic apolipoprotein E-deficient mice.

BACKGROUND: Atherosclerosis and vascular calcification (VC) progression in chronic kidney disease is favored by disturbances of mineral metabolism. We compared the effect of phosphate binder lanthanum (La) carbonate with sevelamer-HCl on atherosclerosis, VC and bone structure and function in mice with chronic renal failure (CRF). METHODS: Apolipoprotein E-deficient (apoE(-/-)) mice were randomized to one non-CRF and three CRF groups, fed with standard diet (one non-CRF and one CRF) or diet supplemented with either 3% lanthanum carbonate (La3%) or 3% sevelamer-HCl (Sev3%). RESULTS: Both La3% and Sev3% supplemented CRF mice displayed a decrease of serum phosphorus, calcification at both intimal and medial aortic sites and atherosclerosis. This was associated with a reduction of plaque Type I collagen expression by both binders and of positive nitrotyrosine staining in response to sevelamer-HCl only. Increased mineral apposition and bone formation rates in unsupplemented CRF mice were reduced by Sev3% but not by La3%. CONCLUSIONS: The beneficial effects of La carbonate and sevelamer-HCl on the progression of VC and atherosclerosis in CRF mice could be mainly due to a decrease in phosphate retention and likewise a reduction of arterial Type I collagen expression. The effect of La carbonate differed from that of sevelamer-HCl in that it did not appear to exert its vascular effects via changes in oxidative stress or bone remodeling in the present model.

Impact of polyunsaturated fatty acids on oxidized low density lipoprotein-induced U937 cell apoptosis.

AIM: Dietary supplements in polyunsaturated fatty acids (PUFA), particularly omega-3, are well known for their beneficial effects in preventing cardiovascular diseases (CVD). The aim of this study was to determine the role of PUFA on the modulation of apoptosis induced by hypochlorous acidoxidized LDL (HOCl-oxLDL) in U937 cells. METHODS: We tested the effect of monocyte cell line U937 supplementation with eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), arachidonic acid (ARA) or oleic acid (OA) on the modulation of HOCl-oxLDL-induced apoptosis. RESULTS: First, we showed the incorporation of fatty acids in the cellular membrane in U937 cells. Then, we showed that both EPA and ARA exerted a pro-apoptotic effect through the intrinsic mitochondrial apoptotic pathway including the dissipation of mitochondrial membrane potential followed by cardiolipin depletion, the downstream activation of caspase-3 and the increase in DNA fragmentation. The pro-apoptotic effect of EPA or ARA was completely blocked in U937/Bcl-2 cells. CONCLUSIONS: A new mechanism of dietary supplements in PUFA with likely consequences in apoptosis could be suggested through the mitochondrial pathway in monocytes.

Vitamin A, E, B12, and folic acid in end-stage renal disease Tunisian patients: status and predictive value for overall mortality and cardiovascular events.

BACKGROUND: Vitamin status and role in end stage renal disease (ESRD) is controversial. This study was aimed at assessing vitamin A, E, B12, and folic acid status in Tunisian ESRD patients and testing their predictive value for overall mortality and cardiovascular events (CVE). METHODS: We examined plasma vitamin A, E, B12, and folic acid in 115 ESRD patients and looked for any correlation with all-cause mortality and CVE after a six year follow-up. Vitamin A and E were determined by HPLC and vitamin B12 and folic acid were determined by enzyme immunoassay. RESULTS: At enrolment, plasma vitamin A was higher in patients than controls, while plasma vitamin B12 was higher in HD patients. No significant differences were observed for plasma vitamin E and folic acid concentrations between patients and controls. Folic acid and vitamin B12 levels were higher in supplemented patients. During the follow-up period, 17 patients were lost, 15 died, and 36 presented a CVE. Survival analysis showed that mortality and/or CVE trend to be lower for high folic acid levels (Log Rank = 0.098). Cox's regression analysis showed that high levels of folic acid are inversely related to all-cause mortality and/or CVE [Hazard ratio (95% confidence interval), 0.255 (0.08 - 0.740); p = 0.012]. CONCLUSIONS: Plasma vitamins A, E, B12, and folic acid concentrations are usually normal in Tunisian ESRD patients. High folic acid levels are associated with fewer CVE and better survival. However, as uremia could be associated with functional vitamin deficiency, maintaining high plasma vitamin levels by adequate nutrition and tolerable supplementation would be beneficial in ESRD patients.

The calcimimetic R-568 retards uremia-enhanced vascular calcification and atherosclerosis in apolipoprotein E deficient (apoE-/-) mice.

OBJECTIVE: Secondary hyperparathyroidism of chronic kidney disease promotes vascular calcification. Calcimimetics reduce serum parathyroid hormone, calcium (Ca), and phosphorus by calcium-sensing receptor (CaR) activation. Here we examined possible effects of the calcimimetic R-568 (R-568) on the progression of aortic calcification and atherosclerosis in apoE(-/-) mice with chronic renal failure (CRF) and the potential implication of aortic smooth muscle cell CaR. METHODS AND RESULTS: ApoE(-/-) mice were assigned to 3 CRF groups and 1 non-CRF group receiving daily gavage with R-568, calcitriol, or vehicle. Serum Ca and phosphorus and parathyroid gland volume of CRF mice were decreased by R-568, whereas elevated serum FGF23 and total cholesterol remained unchanged. Both aortic plaque and non-plaque calcification was lower in R-568 mice, and so was atherosclerotic plaque area fraction. In vitro, R-568 induced a decrease in smooth muscle cell calcification when cultured in high phosphate medium. This decrease was abolished in CaR-SiRNA-transfected cells. CONCLUSIONS: The calcimimetic R-568 delayed the progression of both aortic calcification and atherosclerosis in uremic apoE(-/-) mice. This effect was mediated via a better control of hyperparathyroidism including serum Ca and phosphorus. Direct vascular CaR activation also could have played a role in the observed effects.

Effects of increasing docosahexaenoic acid intake in human healthy volunteers on lymphocyte activation and monocyte apoptosis.

Dietary intake of long-chain n-3 PUFA has been reported to decrease several markers of lymphocyte activation and modulate monocyte susceptibility to apoptosis. However, most human studies examined the combined effect of DHA and EPA using relatively high daily amounts of n-3 PUFA. The present study investigated the effects of increasing doses of DHA added to the regular diet of human healthy volunteers on lymphocyte response to tetradecanoylphorbol acetate plus ionomycin activation, and on monocyte apoptosis induced by oxidized LDL. Eight subjects were supplemented with increasing daily doses of DHA (200, 400, 800, 1600 mg) in a TAG form containing DHA as the only PUFA, for 2 weeks each dose. DHA intake dose-dependently increased the proportion of DHA in mononuclear cell phospholipids, the augmentation being significant after 400 mg DHA/d. The tetradecanoylphorbol acetate plus ionomycin-stimulated IL-2 mRNA level started to increase after ingestion of 400 mg DHA/d, with a maximum after 800 mg intake, and was positively correlated (P < 0.003) with DHA enrichment in cell phospholipids. The treatment of monocytes by oxidized LDL before DHA supplementation drastically reduced mitochondrial membrane potential as compared with native LDL treatment. Oxidized LDL apoptotic effect was significantly attenuated after 400 mg DHA/d and the protective effect was maintained throughout the experiment, although to a lesser extent at higher doses. The present results show that supplementation of the human diet with low DHA dosages improves lymphocyte activability. It also increases monocyte resistance to oxidized LDL-induced apoptosis, which may be beneficial in the prevention of atherosclerosis.

Protective effect of eicosapentaenoic acid on palmitate-induced apoptosis in neonatal cardiomyocytes.

Long chain polyunsaturated fatty acids (PUFAs) play an important role in cardioprotection. These effects have been largely attributed to membrane docosahexaenoic acid. Conversely, saturated fatty acids trigger apoptosis in cardiomyocytes, with modifications of mitochondrial properties including cardiolipin loss, cytochrome c release and caspase-3 activation. The purpose of this study was to investigate the chronic effect of eicosapentaenoic acid (EPA) on mitochondrial apoptosis induced by palmitate treatment and the associated signalling pathways. Confluent cultures of rat neonatal cardiomyocytes were treated for 2 days in media enriched with either EPA or arachidonic acid (AA) and then exposed to palmitate (0.5 mM) to induce apoptosis, in the absence of PUFA supplements. The EPA treatment resulted in significant membrane enrichment in n-3 PUFAs, especially in docosapentaenoic acid (DPA), and a large decrease in AA. Both AA and EPA treatments prevented caspase-3 activation, translocation of Bax to the mitochondria and release of cytochrome c induced by palmitate treatment. Furthermore, EPA, but not AA prevented the loss of mitochondrial cardiolipin due to apoptosis. These results suggest that EPA supplementation is able to protect cardiomyocytes against palmitate-induced apoptosis via an implication of different mitochondrial elements, possibly through its elongation to DPA, which is very efficient in cardiomyocytes.

Dietary n-3 polyunsaturated fatty acids and endothelium dysfunction induced by lysophosphatidylcholine in Syrian hamster aorta.

This study investigated the influence of an eicosapentaenoic acid (EPA)- or a docosahexaenoic acid (DHA)-supplemented diet on the deleterious effects of lysophosphatidylcholine (LPC) on endothelium-dependent vasorelaxation of Golden Syrian hamster thoracic aorta. In a second step, LPC-modulated phospholipase A(2) (PLA(2))-derived ways of relaxation were investigated. Golden Syrian hamsters were fed for 6 weeks with a control diet or an EPA- or DHA-supplemented diet. Aortic fatty acid composition was analyzed by gas chromatography. Aortic rings were incubated for 20 minutes with LPC before constructing cumulative concentration-response curves for acetylcholine (ACh; 3 nmol/L-30 micromol/L) or sodium nitroprusside (3 nmol/L-30 micromol/L). The EPA- or DHA-supplemented diet increased n-3 polyunsaturated fatty acids in aortic fatty acids content because of the increase of EPA or DHA content, respectively, and decreased arachidonic acid aortic content. Lysophosphatidylcholine (1, 10, 15, and 20 micromol/L) induced a concentration-dependent inhibition of ACh-induced relaxation of preconstricted aortic rings in the control group, but did not influence sodium nitroprusside-induced aortic relaxation. The DHA- or EPA-supplemented diet worsened LPC (20 micromol/L) inhibitory effects on ACh-induced vasorelaxation. In the control diet group, ACh-induced relaxation was abolished by the nitric oxide synthase inhibitor (l-N(G)-nitro-arginine methyl ester; 100 micromol/L), whether LPC was added or not. The ACh-induced vasorelaxation was partially inhibited by PLA(2) inhibitors methyl arachidonyl fluorophosphonate (25 micromol/L) and arachidonyl trifluoromethyl ketone (20 micromol/L) as well as by the combination of 2 Ca(2+)-dependent potassium (K(Ca)) channel inhibitors charybdotoxin (0.1 micromol/L) plus apamin (0.3 micromol/L). In the presence of LPC (20 micromol/L), ACh-induced vasorelaxation was abolished by these inhibitors. These effects were not influenced by DHA or EPA diet. Our results suggested that EPA- or DHA-supplemented diet did not exhibit any beneficial effect against LPC-induced inhibition of endothelium-dependent aortic relaxation in Golden Syrian hamsters. These LPC effects were associated in our study not only with an inhibition of nitric oxide-dependent vasorelaxation, but also with a concomitant activation of a compensatory vasorelaxant pathway depending both on PLA(2) metabolites and on K(Ca) channel opening.

Effect of oral calcium carbonate on aortic calcification in apolipoprotein E-deficient (apoE-/-) mice with chronic renal failure.

BACKGROUND: In chronic kidney disease (CKD) patients, the intake of calcium-based phosphate binders is associated with a marked progression of coronary artery and aortic calcification, in contrast to patients receiving calcium-free phosphate binders. The aim of this study was to reexamine the role of calcium carbonate in vascular calcification and to analyse its effect on aortic calcification-related gene expression in chronic renal failure (CRF). METHODS: Mice deficient in apolipoprotein E underwent either sham operation or subtotal nephrectomy to create CRF. They were then randomly assigned to one of the three following groups: a control non-CRF group and a CRF group fed on standard diet, and a CRF group fed on calcium carbonate enriched diet, for a period of 8 weeks. Aortic atherosclerotic plaque and calcification were evaluated using quantitative morphologic image processing. Aortic gene and protein expression was examined using immunohistochemistry and Q-PCR methods. RESULTS: Calcium carbonate supplementation was effective in decreasing serum phosphorus but was associated with a higher serum calcium concentration. Compared with standard diet, calcium carbonate enriched diet unexpectedly induced a significant decrease of both plaque (p<0.05) and non-plaque-associated calcification surface (p<0.05) in CRF mice. It also increased osteopontin (OPN) protein expression in atherosclerotic lesion areas of aortic root. There was also a numerical increase in OPN and osteoprotegerin gene expression in total thoracic aorta but the difference did not reach the level of significance. Finally, calcium carbonate did not change the severity of atherosclerotic lesions. CONCLUSION: In this experimental model of CRF, calcium carbonate supplementation did not accelerate but instead decreased vascular calcification. If our observation can be extrapolated to humans, it appears to question the contention that calcium carbonate supplementation, at least when given in moderate amounts, necessarily enhances vascular calcification. It is also compatible with the hypothesis of a preponderant role of phosphorus over that of calcium in promoting vascular calcification in CRF.

Effects of oral vitamin C supplementation in hemodialysis patients: a proteomic assessment.

Evidence indicates that oxidative stress is present in dialysis patients, and is associated with vitamin C deficiency. Limited data are available regarding the effects of vitamin C supplementation on oxidative stress and inflammation markers in these patients. Moreover, there are no data available on plasma polypeptide fingerprints by proteome analysis before and after vitamin C supplementation. Therefore, we analyzed plasma samples from a prospective, randomized, open-labeled trial to assess the effects of oral vitamin C supplementation (250 mg three times per week), to define the plasma polypeptide pattern in hemodialysis patients. Our results reveal that more than 30 polypeptides show significant changes in the dialysis patients in comparison to controls with normal renal function, and that several polypeptides are affected/normalized by oral vitamin C supplementation. These results underline the remarkable potential for proteomics to recognize specific peptide profiles in different pathological situations, which might not be detected by classical methods.

[Oxidative stress in end stage renal disease: evidence and association with cardiovascular events in Tunisian patients]. / Stress oxydant chez les insuffisants rénaux chroniques: evidence et association avec les maladies cardiovasculaires chez des urémiques Tunisiens.

We assessed oxidative stress in 35 chronic renal failure under conservative treatment (CRF), 50 hemodialysed (HD) and 30 renal transplant (RT) patients, and 31 age- and sex-matched healthy subjects. Compared to controls, CRF patients exhibited significantly higher conjugated dienes (139 +/- 37 versus 121 +/- 22 micromol/l) and LDL oxidation (126 +/- 65 versus 99 +/- 46 micromol/l). Glutathione peroxidase activity was decreased in CRF and HD (5.31 +/- 2.46 and 5.39 +/- 2.32 versus 7.42 +/- 2.72 U/ml in healthy subjects). Superoxide dismutase activity was lower in HD (91 +/- 38 U/ml) and higher in RT patients (132 +/- 33 U/ml) than controls (116 +/- 30 U/ml). Plasma zinc concentrations were significantly decreased in CRF and HD patients and copper concentrations were significantly decreased in TPR. Plasma selenium levels were normal in the three groups of patients. Vitamin A was significantly increased, whereas vitamin E was normal in the 3 groups of patients compared to healthy controls. Total antioxidant status was increased in CRF and HD, but not in RT patients. Patients with cardiovascular disease showed increased serum copper, and significantly decreased glutathione peroxidase activity. This study revealed an oxidative stress in CRF and HD patients that may favour the development of cardiovascular diseases.

Effects of oral vitamin C supplementation on oxidative stress and inflammation status in haemodialysis patients.

BACKGROUND: There is increasing evidence for the presence of oxidative stress and vitamin C deficiency in dialysis patients. Limited data, however, are available regarding the effects of vitamin C supplementation on oxidative stress and inflammation markers in such patients. METHODS: We ran a prospective, randomized, open-label trial to assess the effects of oral vitamin C supplementation (250 mg three times per week) for 2 months on well-defined oxidative and inflammatory markers in 33 chronic haemodialysis (HD) patients. RESULTS: Normalization of plasma total vitamin C and ascorbate levels by oral vitamin C supplementation did not modify plasma levels of carbonyls, C-reactive protein and albumin, or erythrocyte concentrations of reduced and oxidized glutathione. CONCLUSION: Short-term oral vitamin C supplementation did not modify well-defined oxidative/antioxidative stress and inflammation markers in HD patients. Whether a higher oral dose or the intravenous route can modify these markers remains to be determined.

The antioxidant N-acetylcysteine prevents accelerated atherosclerosis in uremic apolipoprotein E knockout mice.

BACKGROUND: Cardiovascular disease is the most frequent cause of mortality in chronic renal failure (CRF). Therefore, it is important to identify appropriate treatment measures. The antioxidant N-acetylcysteine (NAC) has been shown to reduce cardiovascular events in hemodialysis patients. Here we examine a possible direct effect of NAC supplementation on uremia-enhanced atherosclerosis in apolipoprotein E-deficient (apoE(-/-)) mice. METHODS: Uremia was induced surgically in 8-week-old female apoE(-/-) mice. Two weeks after creation of CRF mice were randomized to receive either NAC (daily oral gavage with 200 mg/kg for 8 weeks) or placebo. They were compared to a control group of sham-operated apoE(-/-) mice receiving placebo. After 8 weeks of treatment, the mice were sacrificed, and the cross-section surface area of atherosclerotic plaques was measured in aortic root and descending aorta. RESULTS: At 10 weeks following surgery, atherosclerotic lesions were significantly larger in uremic apoE(-/-) mice than in nonuremic controls. This accelerated atherosclerosis was associated with an increase in aortic nitrotyrosine expression and collagen plaque content. NAC treatment inhibited the progression of atherosclerotic lesions and plaque collagen content compared with placebo treatment. In addition, plaques from NAC-treated uremic animals showed a significant decrease in nitrotyrosine expression whereas the degree of macrophage infiltration was comparable in both uremic groups. There was no difference in mean arterial blood pressure between the three groups. CONCLUSION: We show for the first time that the antioxidant NAC is capable of reducing atheroma progression, in an animal model of uremia-enhanced atherosclerosis, probably via a decrease in oxidative stress.

Effect of dietary docosahexaenoic acid on the endothelium-dependent vasorelaxation in diabetic rats.

1. The aim of the present study was to investigate the responses to acetylcholine (ACh; 3 nmol/L-30 micromol/L) and sodium nitroprusside (SNP; 3 nmol/L-30 micromol/L) of precontracted aortic rings from diabetic rats supplemented with docosahexaenoic acid (DHA). 2. Diabetes was induced by streptozotocin (STZ; 55 mg/kg). Diabetic and sham rats were fed, over a period of 8 weeks, either control diet or a DHA-supplemented diet. Aortic endothelial fatty acid composition was analysed by gas chromatography. The involvement of endothelial-derived nitric oxide (NO) and cyclo-oxygenase (COX) metabolites in response to ACh was assessed using the NO synthase inhibitor N(G)-nitro-L-arginine methyl ester (100 micromol/L) and the COX inhibitor indomethacin (1 micromol/L), respectively. 3. The DHA-supplemented diet induced a small increase in n-3 polyunsaturated fatty acids (PUFA; P < 0.001) owing to the incorporation of DHA in the endothelial cells of sham animals (1.6 +/- 0.2% in the DHA group compared with traces in the control group; P < 0.001) and diabetic animals (1.3 +/- 0.2% in the DHA group compared with traces in control group; P < 0.001), without a decrease in n-6 PUFA, despite a small decrease in arachidonic acid content (P < 0.05). Diabetes did not modify the incorporation of DHA in endothelial cells, but did significantly increase the arachidonic acid content (0.6 +/- 0.0 vs 0.4 +/- 0.1% in control group in the STZ and sham groups, respectively; P < 0.001). Acetylcholine-induced relaxation was significantly reduced in STZ groups compared with the sham groups (P < 0.001) and the DHA-supplemented diet did not modify these effects. In contrast, neither the DHA-supplemented diet nor diabetes affected the aortic relaxation induced by SNP. N(G)-Nitro-L-arginine methyl ester strongly inhibited the relaxant effects of ACh in the sham groups (P < 0.001) and abolished ACh-induced relaxation in the STZ groups (P < 0.001). The diet did not modify these effects. In the presence of indomethacin, the relaxation induced by ACh was decreased in the sham groups (P < 0.01), but not in the STZ groups. The DHA-supplemented diet did not have any effect on these responses. 4. In conclusion, these results suggest that, in the present study, the endothelial dysfunction occurring in the rat model of STZ-induced diabetes is associated with modifications of both the synthesis of COX derivatives and NO metabolism and is not affected by dietary supplementation with DHA.