Plasma type I collagen α1 chain in relation to coronary artery disease: findings from a prospective population-based cohort and an acute myocardial infarction prospective cohort in Sweden.

OBJECTIVES: To investigate the association between type I collagen α1 chain (COL1α1) levels and coronary artery disease (CAD) by using absolute quantification in plasma. Also, to investigate the correlates of COL1α1 to clinical characteristics and circulating markers of collagen metabolism. DESIGN: Life conditions, Stress and Health (LSH) study: prospective cohort study, here with a nested case-control design.Assessing Platelet Activity in Coronary Heart Disease (APACHE) study: prospective cohort study. SETTING: LSH: primary care setting, southeast Sweden.APACHE: cardiology department, university hospital, southeast Sweden. PARTICIPANTS: LSH: 1007 randomly recruited individuals aged 45-69 (50% women). Exclusion criteria was serious disease. After 13 years of follow-up, 86 cases with primary endpoint were identified and sex-matched/age-matched to 184 controls. APACHE: 125 patients with myocardial infarction (MI), 73 with ST-elevation MI and 52 with non-ST-elevation MI. EXCLUSION CRITERIA: Intervention study participation, warfarin treatment and short life expectancy. PRIMARY AND SECONDARY OUTCOME MEASURES: Primary outcome was the association between baseline COL1α1 and first-time major event of CAD, defined as fatal/non-fatal MI or coronary revascularisation after 13 years. Secondary outcomes were the association between the collagen biomarkers PRO-C1 (N-terminal pro-peptide of type I collagen)/C1M (matrix metalloproteinase-mediated degradation of type I collagen) and CAD; temporal change of COL1α1 after acute MI up to 6 months and lastly, correlates between COL1α1 and patient characteristics along with circulating markers of collagen metabolism. RESULTS: COL1α1 levels were associated with CAD, both unadjusted (HR=0.69, 95% CI=0.56 to 0.87) and adjusted (HR=0.55, 95% CI=0.41 to 0.75). PRO-C1 was associated with CAD, unadjusted (HR=0.62, 95% CI=0.47 to 0.82) and adjusted (HR=0.61, 95% CI=0.43 to 0.86), while C1M was not. In patients with MI, COL1α1 remained unchanged up to 6 months. COL1α1 was correlated to PRO-C1, but not to C1M. CONCLUSIONS: Plasma COL1α1 was independently and inversely associated with CAD. Furthermore, COL1α1 appeared to reflect collagen synthesis but not degradation. Future studies are needed to confirm whether COL1α1 is a clinically useful biomarker of CAD.

Complete fatty degeneration of thymus associates with male sex, obesity and loss of circulating naïve CD8+ T cells in a Swedish middle-aged population.

BACKGROUND: Fatty degeneration of thymus (or thymus involution) has long been considered a normal ageing process. However, there is emerging evidence that thymic involution is linked to T cell aging, chronic inflammation and increased morbidity. Other factors, aside from chronological age, have been proposed to affect the involution rate. In the present study, we investigated the imaging characteristics of thymus on computed tomography (CT) in a Swedish middle-aged population. The major aims were to establish the prevalence of fatty degeneration of thymus and to determine its associations with demographic, lifestyle and clinical factors, as well as inflammation, T cell differentiation and thymic output. RESULTS: In total, 1 048 randomly invited individuals (aged 50-64 years, 49% females) were included and thoroughly characterized. CT evaluation of thymus included measurements of attenuation, size and a 4-point scoring system, with scale 0-3 based on the ratio of fat and soft tissue. A majority, 615 (59%) showed complete fatty degeneration, 259 (25%) predominantly fatty attenuation, 105 (10%) half fatty and half soft-tissue attenuation, while 69 (6.6%) presented with a solid thymic gland with predominantly soft-tissue attenuation. Age, male sex, high BMI, abdominal obesity and low dietary intake of fiber were independently associated with complete fatty degeneration of thymus. Also, fatty degeneration of thymus as well as low CT attenuation values were independently related to lower proportion of naïve CD8+ T cells, which in turn was related to lower thymic output, assessed by T-cell receptor excision circle (TREC) levels. CONCLUSION: Among Swedish middle-aged subjects, nearly two-thirds showed complete fatty degeneration of thymus on CT. This was linked to depletion of naïve CD8+ T cells indicating that CT scans of thymus might be used to estimate immunological aging. Furthermore, our findings support the intriguing concept that obesity as well as low fiber intake contribute to immunological aging, thereby raising the possibility of preventive strategies.

The Effects of Dairy and Plant-Based Liquid Components on Lutein Liberation in Spinach Smoothies.

Lutein is a dietary lipophilic compound with anti-inflammatory properties. We have previously shown that dairy fat can improve the lutein content in spinach smoothies. It is, however, unclear whether fat concentrations and fermentation status in dairy products affect lutein liberation in smoothies. Moreover, plant-based milks vary in fat, protein, and fiber content which may affect lutein dissolution. This study aimed to provide translatable information to consumers by comparing lutein liberation in spinach smoothies made with different dairy or plant-based liquids in domestic settings. The smoothies were digested in vitro, and liberated lutein was measured by high-performance liquid chromatography (HPLC). High-fat and medium-fat cow's milk, as well as coconut milk with and without additives, were found to significantly improve lutein liberation by 36%, 30%, 25%, and 42%, respectively, compared to blending spinach with water alone. Adjustment models suggested that the effects of cow's milk and coconut milk were derived from fat and protein, respectively. On the other hand, soymilk with and without additives showed significantly reduced lutein liberation by 40% and 61%, respectively. To summarize, only 4 out of 14 tested liquids increased lutein liberation in spinach smoothies. The results highlight the importance of testing food companions for lipophilic active ingredients.

Oxidative stress response in regulatory and conventional T cells: a comparison between patients with chronic coronary syndrome and healthy subjects.

BACKGROUND: Inflammation and oxidative stress form a vicious circle in atherosclerosis. Oxidative stress can have detrimental effects on T cells. A unique subset of CD4+ T cells, known as regulatory T (Treg) cells, has been associated with atheroprotective effects. Reduced numbers of Treg cells is a consistent finding in patients with chronic coronary syndrome (CCS). However, it is unclear to what extent these cells are sensitive to oxidative stress. In this pilot study, we tested the hypothesis that oxidative stress might be a potential contributor to the Treg cell deficit in CCS patients. METHODS: Thirty patients with CCS and 24 healthy controls were included. Treg (CD4+CD25+CD127-) and conventional T (CD4+CD25-, Tconv) cells were isolated and treated with increasing doses of H2O2. Intracellular ROS levels and cell death were measured after 2 and 18 h, respectively. The expression of antioxidant genes was measured in freshly isolated Treg and Tconv cells. Also, total antioxidant capacity (TAC) was measured in fresh peripheral blood mononuclear cells, and oxidized (ox) LDL/LDL ratios were determined in plasma. RESULTS: At all doses of H2O2, Treg cells accumulated more ROS and exhibited higher rates of death than their Tconv counterparts, p < 0.0001. Treg cells also expressed higher levels of antioxidant genes, including thioredoxin and thioredoxin reductase-1 (p < 0.0001), though without any differences between CCS patients and controls. Tconv cells from CCS patients were, on the other hand, more sensitive to oxidative stress ex vivo and expressed more thioredoxin reductase-1 than Tconv cells from controls, p < 0.05. Also, TAC levels were lower in patients, 0.97 vs 1.53 UAE/100 µg, p = 0.001, while oxLDL/LDL ratios were higher, 29 vs 22, p = 0.006. CONCLUSION: Treg cells isolated from either CCS patients or healthy controls were all highly sensitive to oxidative stress ex vivo. There were signs of oxidant-antioxidant imbalance in CCS patients and we thus assume that oxidative stress may play a role in the reduction of Treg cells in vivo.

Salivary and plasma levels of matrix metalloproteinase-9 and myeloperoxidase at rest and after acute physical exercise in patients with coronary artery disease.

BACKGROUND: Low-grade systemic inflammation is a predictor of recurrent cardiac events in patients with coronary artery disease (CAD). Plasma proteins such as matrix metalloproteinase (MMP)-9 and myeloperoxidase (MPO) have been shown to reflect basal as well as stress-induced inflammation in CAD. Measurements of MMP-9 and MPO in saliva might pose several advantages. Therefore, we investigated whether salivary levels of MMP-9 and MPO corresponded to plasma levels in patients with coronary artery disease (CAD), both at rest and after acute physical exercise. METHODS: A bicycle ergometer test was used as a model for stress-induced inflammation. Twenty-three CAD patients performed the test on two occasions 3-6 months apart. Whole unstimulated saliva was collected before, directly after and 30 min after exercise while plasma was collected before and after 30 min. MMP-9 and MPO in saliva and plasma were determined by Luminex. RESULTS: MMP-9 and MPO levels were 2- to 4-fold higher in saliva than in plasma. Amongst the saliva samples, and also to a great extent amongst the plasma samples, the levels of both types of protein showed strong intercorrelations between the levels at rest and after exercise during the two visits. However, there were no (or weak) correlations between salivary and plasma MMP-9 and none between salivary and plasma MPO. CONCLUSION: We conclude that salivary diagnostics cannot be used to assess systemic levels of MMP-9 and MPO in CAD patients, neither at rest nor after acute physical exercise.

Liberation of lutein from spinach: Effects of heating time, microwave-reheating and liquefaction.

Lutein, abundant in dark leafy vegetables, has been associated with several health promoting effects. Still, to what extent different preparation conditions and practices affect the liberation of lutein from food is not fully understood. Here, we compared a range of domestic methods under realistic conditions to prepare spinach, the most common lutein-rich vegetable. After preparations, samples were processed by in vitro digestion and lutein was quantified by HPLC. Data indicate that short-term and medium-term heating of spinach, independent of heating method, substantially reduced liberated lutein and reduction was most pronounced after long boiling times. Interestingly, the loss of lutein in heated samples was partly compensated when samples were reheated in the microwave. However, the highest yield of liberated lutein was obtained from liquefied spinach. Additional dairy enhanced the liquefaction effect. Thus, for optimal liberation of lutein, liquefaction of raw spinach appears to be the method of choice.

Glucocorticoid sensitivity and inflammatory status of peripheral blood mononuclear cells in patients with coronary artery disease.

OBJECTIVE: Mechanisms behind sustained inflammation in patients with coronary artery disease (CAD) are not clarified but hypothalamus-pituitary-adrenal (HPA) axis dysfunction may have a role. Here, we investigated whether inflammatory status of peripheral blood mononuclear cells (PBMCs) was associated with altered glucocorticoid sensitivity in CAD patients. METHODS: In 55 CAD patients and 30 controls, mRNA levels of GR-α, GR-ß, NF-κB, IκBα, MMP-9 and TIMP-1 were measured in PBMCs. Suppressive effects of dexamethasone on GR-α, GR-ß, NF-κB, IκBα, MMP-9 and TIMP-1 mRNA levels were assessed in PBMCs ex vivo. Salivary cortisol was repeatedly measured over 3 days. RESULTS: GR-α mRNA levels were higher in CAD patients than in controls, 0.50 (0.38-0.59) versus 0.26 (0.18-0.37), p < .001, while GR-ß mRNA levels were equally low in both groups. GR-α mRNA expression was associated with inflammatory gene expression and, also, with flatter diurnal cortisol rhythm. In both patients and controls, dexamethasone suppressed gene expression of NF-κB, IκBα, MMP-9 and TIMP-1 (p < .001). Dexamethasone also reduced GR-α mRNA levels (p < .001), while LPS increased it (p < .001). CONCLUSIONS: PBMCs from CAD patients displayed an inflammatory gene expression profile. This was not explained by reduced glucocorticoid sensitivity. Instead, inflammation was associated with increased expression of GR-α mRNA, suggesting a hypocortisolemic state. Key messages • Peripheral blood mononuclear cells from patients with coronary artery disease (CAD) display an inflammatory gene expression profile. • This inflammatory state cannot be explained by reduced glucocorticoid sensitivity in CAD patients. • Instead, the inflammatory gene expression profile is associated with upregulated levels of glucocorticoid receptor-α mRNA, suggesting a hypocortisolemic state.

Effect of long-term dietary sphingomyelin supplementation on atherosclerosis in mice.

Sphingomyelin (SM) levels in the circulation correlate positively with atherosclerosis burden. SM is a ubiquitous component of human diets, but it is unclear if dietary SM increases circulating SM levels. Dietary choline increases atherosclerosis by raising circulating trimethylamine N-oxide (TMAO) levels in mice and humans. As SM has a choline head group, we ask in this study if dietary SM accelerates atherosclerotic lesion development by increasing circulating SM and TMAO levels. Three studies were performed: (Study 1) C57BL/6 mice were maintained on a high fat diet with or without SM supplementation for 4 weeks prior to quantification of serum TMAO and SM levels; (Study 2) atherosclerosis was studied in apoE-/- mice after 16 weeks of a high fat diet without or with SM supplementation and (Study 3) apoE-/- mice were maintained on a chow diet for 19 weeks without or with SM supplementation and antibiotic treatment prior to quantification of atherosclerotic lesions and serum TMAO and SM levels. SM consumption did not increase circulating SM levels or atherosclerosis in high fat-fed apoE-/- mice. Serum TMAO levels in C57BL/6 mice were low and had no effect atherosclerosis lesion development. Dietary SM supplementation significantly reduced atherosclerotic lesion area in the aortic arch of chow-fed apoE-/- mice. This study establishes that dietary SM does not affect circulating SM levels or increase atherosclerosis in high fat-fed apoE-/- mice, but it is anti-atherogenic in chow-fed apoE-/- mice.

Lutein exerts anti-inflammatory effects in patients with coronary artery disease.

BACKGROUND AND AIMS: Many coronary artery disease (CAD) patients exhibit chronic low-grade inflammation. Carotenoids are anti-oxidants with potential anti-inflammatory properties. Here, we first assessed relationships between interleukin (IL)-6 and individual carotenoids in plasma from CAD patients. Based on the results, we proceeded to assess anti-inflammatory effects of one carotenoid, lutein, in peripheral blood mononuclear cells (PBMCs) from CAD patients. METHODS: Lutein + zeaxanthin (isomers with lutein being dominant), ß-cryptoxanthin, lycopene, α- and ß-carotene and IL-6 were measured in plasma from 134 patients with stable angina (SA) and 59 patients with acute coronary syndrome. In 42 patients, plasma measurements were also performed 3 months after coronary intervention. PBMCs from SA patients were pre-treated with lutein (1, 5 and 25 µM) for 24 h followed by 24 h incubation ± lipopolysaccharide (LPS). Cell pellets were collected for IL-6, IL-1ß and TNF mRNA and intracellular lutein. Cytokine secretion was measured in cell media. RESULTS: Only lutein + zeaxanthin were inversely correlated with IL-6 in SA patients at baseline (r = -0.366, p < 0.001) and follow-up (r = -0.546, p < 0.001). Ex vivo, lutein was taken up by PBMCs from SA patients in a dose- and time-dependent manner. Pre-treatment with lutein dose-dependently lowered LPS-induced secretion of IL-6, IL-1ß (p < 0.01) and TNF (p < 0.05), and also reduced IL-6, IL-1ß and TNF mRNA expression (p < 0.05). CONCLUSIONS: Clinical findings highlighted the inverse association between lutein and IL-6 in CAD patients. Anti-inflammatory effects of lutein in PBMCs from CAD patients were consolidated in ex vivo experiments. Taken together, these results show that lutein has the potential to play a role in resolution of chronic inflammation in CAD patients.

Dietary sphingomyelin lowers hepatic lipid levels and inhibits intestinal cholesterol absorption in high-fat-fed mice.

Controlling intestinal lipid absorption is an important strategy for maintaining lipid homeostasis. Accumulation of lipids in the liver is a major risk factor for metabolic syndrome and nonalcoholic fatty liver disease. It is well-known that sphingomyelin (SM) can inhibit intestinal cholesterol absorption. It is, however, unclear if dietary SM also lowers liver lipid levels. In the present study (i) the effect of pure dietary egg SM on hepatic lipid metabolism and intestinal cholesterol absorption was measured with [(14)C]cholesterol and [(3)H]sitostanol in male C57BL/6 mice fed a high-fat (HF) diet with or without 0.6% wt/wt SM for 18 days; and (ii) hepatic lipid levels and gene expression were determined in mice given a HF diet with or without egg SM (0.3, 0.6 or 1.2% wt/wt) for 4 weeks. Mice supplemented with SM (0.6% wt/wt) had significantly increased fecal lipid and cholesterol output and reduced hepatic [(14)C]cholesterol levels after 18 days. Relative to HF-fed mice, SM-supplemented HF-fed mice had significantly lower intestinal cholesterol absorption (-30%). Liver weight was significantly lower in the 1.2% wt/wt SM-supplemented mice (-18%). Total liver lipid (mg/organ) was significantly reduced in the SM-supplemented mice (-33% and -40% in 0.6% wt/wt and 1.2% wt/wt SM, respectively), as were triglyceride and cholesterol levels. The reduction in liver triglycerides was due to inactivation of the LXR-SREBP-1c pathway. In conclusion, dietary egg SM has pronounced hepatic lipid-lowering properties in mice maintained on an obesogenic diet.

Hydrogenated phosphatidylcholine supplementation reduces hepatic lipid levels in mice fed a high-fat diet.

The ability of the fatty acid composition of dietary phosphatidylcholine (PC) to affect hepatic lipid levels was investigated in C57BL/6 mice (n=8-10 per group) by feeding: (1) a high-fat semi-purified diet (HF), (2) HF diet supplemented with 1.25 wt% soy PC (SPC), (3) HF with 1.25 wt% hydrogenated soy PC (SPCH), (4) HF with 1.25 wt% egg PC (EPC), and (5) HF with 1.25 wt% hydrogenated egg PC (EPCH). The polyunsaturated fatty acid content (C18:2+C18:3+C20:4) of soy, egg and hydrogenated PC was 70%, 20% and 0%, respectively. Total liver lipid was significantly lower in SPCH and EPCH vs. HF (8.7 ± 0.1 and 8.5 ± 0.5 vs. 11.8 ± 0.6g/100, P<0.05), but not in SPC or EPC. SPCH and EPCH had significantly lower levels of hepatic cholesterol (-52% and -53% vs. HF, respectively). Bioactive lipids (i.e., sphingomyelin and ceramide) were also lower in the liver of SPCH and EPCH rather than in SPC or EPC. Hepatic expression of genes controlling fatty acid synthesis and catabolism were not significantly affected by dietary PC. However, hepatic expression of HMGCR, LDLR and SREBP2 was higher and that of ABCA1, ABCG5 and ABCG8 was reduced in SPCH and EPCH vs. HF. These results demonstrate that hydrogenated PC supplementation reduces hepatic lipid levels in mice fed a high-fat diet supporting the concept that the ability of dietary PC to lower hepatic lipid levels is not due to its content of polyunsaturated fatty acids.

Reduction in intestinal cholesterol absorption by various food components: mechanisms and implications.

A number of different food components are known to reduce plasma and LDL-cholesterol levels by affecting intestinal cholesterol absorption. They include: soluble fibers, phytosterols, saponins, phospholipids, soy protein and stearic acid. These compounds inhibit cholesterol absorption by affecting cholesterol solubilization in the intestinal lumen, interfering with diffusion of luminal cholesterol to the gut epithelium and/or inhibiting molecular mechanisms responsible for cholesterol uptake by the enterocyte. Cholesterol content of intestinal chylomicrons is subsequently reduced, less cholesterol is transported to the liver within chylomicron remnants, hepatic LDL-receptor activity is increased and plasma levels of LDL-cholesterol are decreased. Reduced hepatic VLDL production and less conversion of VLDL to LDL also contribute to lower LDL levels. Certain food components may also affect intestinal bile acid metabolism. Further investigation of the way in which these functional ingredients affect intestinal lipid metabolism will facilitate their use and application as cardiovascular nutraceuticals.

Dietary phospholipids and intestinal cholesterol absorption.

Experiments carried out with cultured cells and in experimental animals have consistently shown that phospholipids (PLs) can inhibit intestinal cholesterol absorption. Limited evidence from clinical studies suggests that dietary PL supplementation has a similar effect in man. A number of biological mechanisms have been proposed in order to explain how PL in the gut lumen is able to affect cholesterol uptake by the gut mucosa. Further research is however required to establish whether the ability of PLs to inhibit cholesterol absorption is of therapeutic benefit.

Dietary krill oil supplementation reduces hepatic steatosis, glycemia, and hypercholesterolemia in high-fat-fed mice.

Krill oil (KO) is rich in n-3 fatty acids that are present in phospholipids rather than in triglycerides. In the present study, we investigated the effects of dietary KO on cardiometabolic risk factors in male C57BL/6 mice fed a high-fat diet. Mice (n = 6-10 per group) were fed for 8 weeks either: (1) a nonpurified chow diet (N); (2) a high-fat semipurified diet containing 21 wt % buttermilk + 0.15 wt % cholesterol (HF); (3) HF supplemented with 1.25 wt % KO (HFKO1.25); (4) HF with 2.5 wt % KO (HFKO2.5); or (5) HF with 5 wt % KO (HFKO5.0). Dietary KO supplementation caused a significant reduction in liver wt (i.e., hepatomegaly) and total liver fat (i.e., hepatic steatosis), due to a dose-dependent reduction in hepatic triglyceride (mean +/- SEM: 35 +/- 6, 47 +/- 4, and 51 +/- 5% for HFKO1.25, -2.5, and -5.0 vs HF, respectively, P < 0.001) and cholesterol (55 +/- 5, 66 +/- 3, and 71 +/- 3%, P < 0.001). Serum cholesterol levels were reduced by 20 +/- 3, 29 +/- 4, and 29 +/- 5%, and blood glucose was reduced by 36 +/- 5, 34 +/- 6, and 42 +/- 6%, respectively. Serum adiponectin was increased in KO-fed animals (HF vs HFKO5.0: 5.0 +/- 0.2 vs 7.5 +/- 0.6 microg/mL, P < 0.01). These results demonstrate that dietary KO is effective in improving metabolic parameters in mice fed a high-fat diet, suggesting that KO may be of therapeutic value in patients with the metabolic syndrome and/or nonalcoholic fatty liver disease.

Dietary phospholipid-rich dairy milk extract reduces hepatomegaly, hepatic steatosis and hyperlipidemia in mice fed a high-fat diet.

Recent studies have suggested that milk and certain dairy food components have the potential to protect against cardiovascular disease. In order to determine whether the addition of milk-derived phospholipids to the diet results in an improvement in metabolic and cardiovascular risk factors, we studied four groups (n=10) of C57BL/6 mice that were fed: (1) a normal non-purified diet (N); (2) the normal non-purified diet supplemented with phospholipid-rich dairy milk extract (PLRDME, 2.5% by wt) (NPL); (3) a high-fat semi-purified diet (HF) containing 21% butterfat+0.15% cholesterol by wt; or (4) HF supplemented with 2.5% by wt PLRDME (HFPL). Dietary PLRDME supplementation did not have a significant effect on metabolic parameters in mice fed the N diet. In contrast, in high-fat fed mice, PLRDME caused a significant decrease in: (a) liver wt (1.57+/-0.06 g vs. 1.20+/-0.04 g, P<0.001), (b) total liver lipid (255+/-22 mg vs. 127+/-13 mg, P<0.001, (c) liver triglyceride (TG) and total cholesterol (TC) 236+/-25 micromol/g vs. 130+/-8 micromol/g (P<0.01), 40+/-7 micromol/g vs. 21+/-2 micromol/g (P<0.05), respectively); and serum lipids (TG: 1.4+/-0.1 mmol/L vs. 1.1+/-0.1 mmol/L, P=0.01; TC: 4.6+/-0.2 mmol/L vs. 3.6+/-0.2 mmol/L, P<0.001; and PL: 3.3+/-0.1 mmol/L vs. 2.6+/-0.1 mmol/L, P<0.01). These data indicate that dietary PLRDME has a beneficial effect on hepatomegaly, hepatic steatosis and elevated serum lipid levels in mice fed a high-fat diet, providing evidence that PLRDME might be of therapeutic value in human subjects as a hepatoprotective or cardioprotective nutraceutical.