Marit S. Nenseter

Very long chain n-3 and n-6 polyunsaturated fatty acids inhibit proliferation of human T-lymphocytes in vitro.

Abstract. The effect of marine n‐3 polyunsaturated fatty acids on proliferation of human T‐cells in vitro was compared to other polyunsaturated, monounsa‐turated and saturated fatty acids. Monoenes and saturated fatty acids had little effect on T‐cell proliferation. Eicosapentaenoic acid and docosahexaenoic acid exerted a strong dose‐dependent inhibitory effect on proliferation of mitogen‐ or antigen‐stimulated T‐cells, similar to that observed for arachidonic acid. Sixty μM of albumin‐bound eicosapentaenoic acid or arachidonic acid promoted 25–40% inhibition of proliferation of T‐cells stimulated with mitogen, whereas the same concentration of albumin‐bound docosahexaenoic acid promoted 60% inhibition. When epidermal cells (Langerhans cells) were used as antigen‐presenting cells, 100 μM of albumin‐bound eicosapentaenoic acid or arachidonic acid caused 40% inhibition on T‐cell proliferation. Low density lipo‐protein (LDL), isolated after four months of dietary intake of fish oil or corn oil, inhibited mitogen‐stimulated T‐cell proliferation in a dose‐dependent manner. Fish oil‐ and corn oil‐enriched LDL showed similar ability to inhibit T‐cell proliferation. Epidermal cells preincubated with docosahexaenoic acid, and extensively washed before adding purified T‐cells and antigen, resulted in a strong inhibition of T‐cell proliferation, whereas preincubation of purified T‐cells with docosahexaenoic acid did not cause any inhibitory effect. Cyclooxygenase and lipoxygenase inhibitors (indomethacin, acetylsalicyclic acid, nordi‐hydroguaertic acid) did not affect the antiproliferative effect of eicosapentaenoic acid and arachidonic acid, neither did the antioxidants butylated hydroxytoluene or alpha‐tocopherol. Eicosanoids, (PCE2, PGE3, LTB4, LTB5 and lipoxin A or lipoxin B) added directly to mitogen‐stimulated peripheral blood mononuclear cells (PBMC) did not influence T‐cell proliferation significantly. Decreased viability was observed when mitogen‐stimulated lymphocytes were cultured with essential polyunsaturated fatty acids, whereas the viability of unstimulated lymphocytes was hardly influenced by the same fatty acids. We conclude that; (a) pharmacological albumin‐bound concentrations of the highly unsaturated fatty acids eicosapentaenoic acid and docosahexaenoic acid promote a strong antiproliferative effect on mitogen‐ and antigen‐stimulated human T‐cells: (b) docosahexaenoic acid can suppress accessory cell function and consequently suppress T‐cell activation; (c) physiologic concentration of LDL particles rich in n‐3 and n‐6 fatty acids, both promote a dose‐dependent antiproliferative effect on mitogen‐stimulated PBMC; (d) the inhibition is independent of eicosanoid metabolites: and (e) lipid peroxidation seems unlikely to be responsible for the antiproliferative effect.

DIETARY POLYUNSATURATES AND PEROXIDATION OF LOW DENSITY LIPOPROTEIN

Oxidative modification of low density lipoprotein is influenced by dietary polyunsaturates. Omega-6 polyunsaturated fatty acids enhance the susceptibility of low density lipoprotein to oxidation compared with monoenes. Most studies on omega-3 fatty acids also exhibit increased peroxidation of low density lipoprotein, although these data are more conflicting. Future studies should focus on additional information concerning dietary intake of antioxidants, fatty acids and lipid peroxides, as well as on the importance of low density lipoprotein oxidation in vivo.

Folic acid treatment reduces elevated plasma levels of asymmetric dimethylarginine in hyperhomocysteinaemic subjects.

Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthase, has been suggested to be a novel risk factor for endothelial dysfunction. It has previously been reported that hyperhomocysteinaemia may be associated with impaired endothelium-dependent vasodilation and reduced plasma level of NO-derived endproducts (NOx). In the present study, plasma levels of arginine and ADMA were measured in twenty-one healthy control subjects, and in twenty-one hyperhomocysteinaemic subjects before and after 6 weeks and 12 months of folic acid supplementation, and compared with previously measured plasma NOx values in the hyperhomocysteinaemic subjects. Compared with control subjects, hyperhomocysteinaemic subjects had higher plasma levels of arginine and ADMA. More importantly, folic acid therapy significantly reduced plasma levels of arginine and ADMA. Furthermore, plasma levels of arginine and ADMA were positively correlated with plasma homocysteine levels and negatively correlated with plasma folate, as well as negatively correlated with plasma NOx. Our results suggest that ADMA may be a mediator of the atherogenic effects of homocysteine.

Plasma homocysteine concentration related to diet, endothelial function and mononuclear cell gene expression among male hyperlipidaemic smokers.

Elevated plasma concentration of homocysteine is an independent risk factor for development of cardiovascular diseases.

Folic Acid Treatment Reduces Chemokine Release From Peripheral Blood Mononuclear Cells in Hyperhomocysteinemic Subjects

Elevated plasma homocysteine concentration is an independent risk factor for cardiovascular disease. However, the mechanisms by which hyperhomocysteinemia induces vascular disease are uncertain. An early step in atherogenesis involves leukocyte migration into the arterial wall, a process regulated in part by chemokines. We hypothesized that homocysteine may exert its atherogenic effect in part through chemokine-mediated mechanisms, and in the present study, we examined the effects of folic acid supplementation for 6 weeks on chemokine levels in hyperhomocysteinemic individuals. Data showed the following: (1) Compared with control subjects, hyperhomocysteinemic subjects had elevated plasma levels of the CXC chemokines, epithelial neutrophil-activating peptide (ENA)-78 (P <0.05), and growth-regulated oncogene (GRO)&agr; (P =0.088), and homocysteine was significantly correlated with ENA-78 and GRO&agr;. (2) During folic acid treatment, normalization of homocysteine levels was accompanied by a marked reduction in oxidized low density lipoprotein–stimulated release of CXC chemokines (ie, GRO&agr;, ENA-78, and interleukin-8) and CC chemokines (ie, monocyte chemoattractant peptide-1 and RANTES) in peripheral blood mononuclear cells from these individuals. (3) The oxidized low density lipoprotein–induced release of ENA-78 from peripheral blood mononuclear cells from control subjects was significantly reduced when cells were incubated in the presence of folic acid. These data may suggest that homocysteine exerts atherogenic effects in part by enhancing chemokine responses in cells involved in atherogenesis and that folic acid supplementation may downregulate these inflammatory responses.

Effect of folic acid treatment on endothelium-dependent vasodilation and nitric oxide-derived end products in hyperhomocysteinemic subjects.

PURPOSE An elevated plasma homocysteine concentration is an independent risk factor for cardiovascular diseases. In this study, we tested the hypothesis that hyperhomocysteinemia induces endothelial dysfunction mediated, at least in part, through nitric oxide-dependent mechanisms and that folic acid supplementation improves endothelial function in hyperhomocysteinemic subjects. SUBJECTS AND METHODS Endothelial function was evaluated in healthy controls and hyperhomocysteinemic subjects by measuring plasma levels of the nitric oxide-derived end products nitrite and nitrate and by assessing vasodilatory responses in the skin microcirculation and forearm vasculature. In the subjects with hyperhomocysteinemia, these measurements were repeated after 6 weeks and 12 months of folic acid supplementation. RESULTS Compared with healthy controls, hyperhomocysteinemic subjects had significantly lower median plasma levels of nitric oxide-derived end products (12.1 microM [range 4.4 to 41.8] versus 24.6 microM [13.6 to 53.2]; P <0.001), a significantly lower endothelium-dependent vasodilatory response to acetylcholine (P <0.01), hyperemic response in the microcirculation (P <0.01), and total forearm blood flow during reactive hyperemia (P = 0.01). There was no significant difference in the endothelium-independent response. Folic acid treatment for 12 months increased the plasma level of nitric oxide-derived end products by 121% (95% confidence interval [CI], 72% to 170%), the vasodilatory response to acetylcholine by 124% (95% CI, 36% to 212%), and the ischemia-mediated hyperemic responses in the microcirculation by 60% (95% CI, 25% to 96%) and in the forearm vasculature by 47% (95% CI, 21% to 73%). CONCLUSIONS Homocysteine appears to induce its atherogenic effect, at least in part, by depressing endothelial function, possibly through nitric oxide-dependent mechanisms. This effect can be reversed by folic acid supplementation.

Increased levels of C-reactive protein and interleukin-6 in hyperhomocysteinemic subjects.

Objective. Elevated plasma homocysteine concentration is considered to be an independent risk factor for cardiovascular disease. However, the mechanisms by which hyperhomocysteinemia are related to vascular disease are unclear. High‐sensitivity C‐reactive protein (CRP), a marker of inflammation, has been reported to be an independent predictor of future myocardial infarction among clinically healthy individuals. Interleukin (IL)‐6 is a regulator of CRP and has a key role in initiation of inflammation. The aim of this study was to investigate whether individuals with increased plasma homocysteine concentrations have altered levels of serum CRP and IL‐6. Material and methods. Serum concentrations of CRP and IL‐6 were measured in 39 individuals with hyperhomocysteinemia and in 39 control subjects matched for gender, age and body mass index (BMI). In addition, the inflammatory effect of IL‐6 on peripheral blood mononuclear cells was measured. Results. Compared to controls, hyperhomocysteinemic subjects have elevated serum levels of CRP and IL‐6 (p⩽0.001 and p<0.005, respectively). Importantly, this raised level of IL‐6 was also seen in hyperhomocysteinemic individuals without accompanying hypercholesterolemia or cardiovascular disease. IL‐6 increased the release of monocyte chemoattractant protein‐1 from peripheral blood mononuclear cells, with particularly enhancing effects in cells from patients with hyperhomocysteinemia. Conclusions. These data suggest that enhanced inflammation may be associated with homocysteine‐related cardiovascular disease, possibly involving IL‐6‐related mechanisms.

CXC-chemokines in coronary artery disease: possible pathogenic role of interactions between oxidized low-density lipoprotein, platelets and peripheral blood mononuclear cells

Summary.  CXC‐chemokines may be involved in atherogenesis. Herein we examined the possible role of CXC‐chemokines in the inflammatory interactions between oxidized (ox‐) low‐density lipoprotein (LDL), platelets and peripheral blood mononuclear cells (PBMC) in 15 patients with coronary artery disease (CAD) without ‘traditional’ risk factors and 15 carefully matched controls. Our main findings were: (a) ox‐LDL stimulated the release of the CXC‐chemokines interleukin (IL)‐8, ENA‐78 and GRO‐α from PBMC, particularly in CAD. (b) In platelets, ox‐LDL induced release of ENA‐78 and, when combined with SFLLRN, also of GRO‐α, with significantly higher response in CAD. (c) Platelet‐rich plasma, especially when costimulated with ox‐LDL, enhanced the release of IL‐8 from PBMC, particularly in CAD patients. (d) Freshly isolated PBMC showed markedly increased IL‐8 mRNA expression in CAD patients. Our findings suggest enhanced inflammatory interactions between ox‐LDL, platelets and PBMC in CAD patients involving CXC‐chemokine related mechanisms, possible contributing to atherogenesis in these and other CAD patients.

Lipoprotein(a) levels in coronary heart disease-susceptible and -resistant patients with familial hypercholesterolemia.

OBJECTIVE Familial hypercholesterolemia (FH) is caused by defects in genes coding for proteins involved in low density lipoprotein (LDL) metabolism, and is associated with increased risk of premature coronary heart disease (CHD). The clinical phenotype of FH exhibits marked variability due to additional metabolic and environmental factors, and further biomarkers are required for appropriate risk assessment. The aim of the present study was to search for risk markers among FH patients. METHODS AND RESULTS Clinical and biochemical parameters of FH subjects with early CHD events (CHD-susceptible) and FH subjects with late or no CHD events (CHD-resistant) were compared. Our data show that CHD-susceptible FH patients had significantly higher Lipoprotein (Lp) (a) levels compared to CHD-resistant FH patients. When subdividing by gender, the main findings were that (i) CHD-susceptible women had significantly higher levels of both Lp(a), low density lipoprotein (LDL) cholesterol and apolipoprotein (apo) B as compared to CHD-resistant women, and (ii) CHD-resistant women had significantly lower Lp(a) levels and higher high density lipoprotein (HDL) cholesterol and apoA-I levels compared to CHD-resistant men. CONCLUSIONS The data suggest that Lp(a) may be an important coronary risk marker in FH patients, in particular in combination with elevated LDL cholesterol levels among female subjects. Thus, measurement of Lp(a) levels may help identifying high-risk individuals who could benefit from an aggressive therapy, including statins to reduce LDL-cholesterol to guideline-recommended levels.

Patients with familial hypercholesterolaemia show enhanced spontaneous chemokine release from peripheral blood mononuclear cells ex vivo. Dependency of xanthomas/xanthelasms, smoking and gender.

AIMS Familial hypercholesterolaemia (FH) is associated with increased risk of premature atherosclerosis and coronary artery disease (CAD). However, onset of clinically manifested CAD varies widely among patients with heterozygous FH, and we hypothesized that inflammatory mediators such as chemokines could contribute to atherogenesis in these patients. METHODS AND RESULTS We compared peripheral blood mononuclear cells (PBMCs) from FH patients with an identical mutation with PBMCs from sex- and age-matched healthy controls with respect to spontaneous and oxidized low density lipoprotein (oxLDL)-stimulated release of chemokines. Our main findings were: (1) PBMCs from FH patients spontaneously released significantly higher levels of macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and interleukin (IL)-8, and had a significantly lower oxLDL-stimulatory ratio for MIP-1alpha and MIP-1beta than cells from healthy controls. (2) Spontaneous release of these chemokines correlated positively and stimulatory ratio correlated negatively with plasma concentrations of total and LDL cholesterol. (3) Among FH patients, release of MIP-1alpha, MIP-1beta and IL-8 from PBMCs varied with the presence of xanthomas/xanthelasms, smoking and gender. (4) In vitro studies showed that FH serum but not control serum was able to induce enhanced spontaneous release of chemokines in PBMCs from both FH patients and control subjects. CONCLUSIONS Our data may suggest that a pathophysiological consequence of FH is enhanced chemokine responses, which in turn may promote recruitment and activation of leukocytes within the vessel wall, contributing to atherosclerosis as well as to the different phenotypes in these patients with an identical FH mutation.