Common Histological Features Suggesting Enchondral Ossification Pathways in Calciphylaxis of Various Origins: A Study of Human Subcutaneous Tissue Biopsies.

Calciphylaxis is a rare, yet underdiagnosed condition causing high mortality in patients with severe renal and cardiovascular disease. Since knowledge of the pathophysiology of calciphylaxis is limited, a differential analysis of histological alterations in patient subgroups with various comorbidities might expose different disease phenotypes and allow deeper insights into the pathophysiology of the condition. Histological markers of osteogenesis and calcification were investigated in a group of 18 patients with clinically and histologically verified calciphylaxis, using immunohistochemical staining. Analysis of staining intensity and distribution of marker proteins in histological structures was performed to evaluate distinct patterns between subgroups with different clinical comorbidities in comparison with a control group. In all cases, immunohistochemical staining for bone matrix proteins, bone-morphogenic proteins and matrix-Gla proteins co-localized with subcutaneous vascular and interstitial calcifications. Significant expression of bone-morphogenic protein-7 and active matrix-Gla protein was observed. Mortality was associated with renal comorbidities and increased expression of bone-morphogenic protein-7. However, no distinct histological patterns were found between subgroups with renal disease, warfarin intake or coexisting micro- and macro-angiopathies. The upregulation of osteogenic markers (including bone-morphogenic protein-7) plays a major role in the development of calciphylaxis. Clinical outcome correlates with kidney function and phosphate handling, suggesting different pathophysiological mechanisms. However, biopsy  at late-stage disease shows a common histological phenotype, involving enchondral ossification.

Yogurt drink fortified with menaquinone-7 improves vitamin K status in a healthy population.

Population-based studies have shown an inverse association between dietary menaquinones (MK-n, vitamin K2) intake, coronary calcification and CHD risk, suggesting a potential role of vitamin K in vascular health. To date, the effects of increased menaquinone intake on (markers of) vascular health have been investigated using predominantly food supplements. Dairy products contain many essential nutrients and can serve as a good matrix for food fortification in order to support health. We were therefore interested to study the effects of a menaquinone-fortified yogurt drink (menaquinone as menaquinone-7 (MK-7); 28 µg MK-7/yogurt drink) on vitamin K status and markers of vascular health. The yogurt drink was also fortified with n-3 PUFA, vitamin D, vitamin C, Ca and Mg to support vascular and/or general health. Healthy men (n 32) and postmenopausal women (n 28) with a mean age of 56 (sd 5) years received either basic or fortified yogurt drink twice per d for 12 weeks. MK-7 was efficiently absorbed from the fortified yogurt drink. Levels of circulating MK-7 were significantly increased from 0·28 to 1·94 ng/ml. In accordance, intake of the fortified yogurt drink improved vitamin K status, as measured by significant decreases in uncarboxylated osteocalcin and desphospho-uncarboxylated matrix Gla-protein. No effects were, however, seen on markers of inflammation, endothelial dysfunction and lipid metabolism. In summary, consumption of a yogurt drink fortified with low doses of among others MK-7 for 3 months significantly improved vitamin K status in a healthy population.

Gla-rich protein is a potential new vitamin K target in cancer: evidences for a direct GRP-mineral interaction.

Gla-rich protein (GRP) was described in sturgeon as a new vitamin-K-dependent protein (VKDP) with a high density of Gla residues and associated with ectopic calcifications in humans. Although VKDPs function has been related with γ-carboxylation, the Gla status of GRP in humans is still unknown. Here, we investigated the expression of recently identified GRP spliced transcripts, the γ-carboxylation status, and its association with ectopic calcifications, in skin basal cell and breast carcinomas. GRP-F1 was identified as the predominant splice variant expressed in healthy and cancer tissues. Patterns of γ-carboxylated GRP (cGRP)/undercarboxylated GRP (ucGRP) accumulation in healthy and cancer tissues were determined by immunohistochemistry, using newly developed conformation-specific antibodies. Both GRP protein forms were found colocalized in healthy tissues, while ucGRP was the predominant form associated with tumor cells. Both cGRP and ucGRP found at sites of microcalcifications were shown to have in vitro calcium mineral-binding capacity. The decreased levels of cGRP and predominance of ucGRP in tumor cells suggest that GRP may represent a new target for the anticancer potential of vitamin K. Also, the direct interaction of cGRP and ucGRP with BCP crystals provides a possible mechanism explaining GRP association with pathological mineralization.

Desphospho-uncarboxylated matrix Gla-protein is associated with mortality risk in patients with chronic stable vascular disease.

BACKGROUND: Vitamin K is the essential co-factor for activation of matrix Gla-protein (MGP), the natural inhibitor of tissue calcification. Biologically inactive, desphospho-uncarboxylated MGP (dp-ucMGP) is a marker of vascular vitamin K status and is described to predict mortality in patients with heart failure and aortic stenosis. We hypothesized that increased dp-ucMGP might be associated with mortality risk in clinically stable patients with chronic vascular disease. MATERIALS AND METHODS: We examined 799 patients (mean age 65.1 ± 9.3 years) who suffered from myocardial infarction, coronary revascularization or first ischemic stroke (pooled Czech samples of EUROASPIRE III and EUROASPIRE-stroke surveys), and followed them in a prospective cohort study. To estimate the 5-year all-cause and cardiovascular mortality we ascertained vital status and declared cause of death. Circulating dp-ucMGP and desphospho-carboxylated MGP (dp-cMGP) were measured by ELISA methods (IDS and VitaK). RESULTS: During a median follow-up of 2050 days (5.6 years) 159 patients died. In the fully adjusted multivariate Cox proportional hazard model, the patients in the highest quartile of dp-ucMGP (≥ 977 pmol/L) had higher risk of all-cause and cardiovascular 5-year mortality [HRR 1.89 (95% CI, 1.32-2.72) and 1.88 (95% CI, 1.22-2.90)], respectively. Corresponding HRR for dp-cMGP were 1.76 (95% CI, 1.18-2.61) and 1.79 (95% CI, 1.12-2.57). CONCLUSIONS: In patients with overt vascular disease, circulating dp-ucMGP and dp-cMGP were independently associated with the risk of all-cause and cardiovascular mortality. Since published results are conflicting regarding the dp-cMGP, we propose only circulating dp-ucMGP as a potential biomarker for assessment of additive cardiovascular risk.

Insights into the association of Gla-rich protein and osteoarthritis, novel splice variants and γ-carboxylation status.

SCOPE: Gla-rich protein (GRP) is a vitamin K dependent protein, characterized by a high density of γ-carboxylated Glu residues, shown to accumulate in mouse and sturgeon cartilage and at sites of skin and vascular calcification in humans. Therefore, we investigated the involvement of GRP in pathological calcification in osteoarthritis (OA). METHODS AND RESULTS: Comparative analysis of GRP patterning at transcriptional and translational levels was performed between controls and OA patients. Using a RT-PCR strategy we unveiled two novel splice variants in human-GRP-F5 and F6-potentially characterized by the loss of full γ-carboxylation and secretion functional motifs. GRP-F1 is shown to be the predominant splice variant expressed in mouse and human adult tissues, particularly in OA cartilage, while an overexpressing human cell model points it as the major γ-carboxylated isoform. Using validated conformational antibodies detecting carboxylated or undercarboxylated GRP (c/uc GRP), we have demonstrated cGRP accumulation in controls, whereas ucGRP was the predominant form in OA-affected tissues, colocalizing at sites of ectopic calcification. CONCLUSION: Overall, our results indicate the predominance of GRP-F1, and a clear association of ucGRP with OA cartilage and synovial membrane. Levels of vitamin K should be further assessed in these patients to determine its potential therapeutic use as a supplement in OA treatment.

Vascular calcification in patients with type 2 diabetes: the involvement of matrix Gla protein.

BACKGROUND: Matrix Gla protein (MGP) is an important inhibitor of calcification. The objective of the present study of patients with type 2 diabetes and normal or slightly altered kidney function was to evaluate levels of inactive, dephospho-uncarboxylated MGP(dp-ucMGP) and total uncarboxylated MGP(t-ucMGP) and assess their links with biological and clinical parameters (including peripheral vascular calcification). METHODS: The DIACART study is a cross-sectional cohort study of 198 patients with type 2 diabetes and normal or slightly altered kidney function. Matrix Gla protein levels were measured with an ELISA and all patients underwent multislice spiral computed tomography scans to score below-knee arterial calcification. RESULTS: In the study population as a whole, the mean dp-ucMGP and t-ucMGP levels were 627 ± 451 pM and 4868 ± 1613 nM, respectively. Glomerular filtration rate, age and current vitamin K antagonist use were independently associated with dp-ucMGP levels. When the study population was divided according to the median peripheral arterial calcification score, patients with the higher score displayed significantly lower t-ucMGP and significantly higher dp-ucMGP levels. Furthermore, plasma dp-ucMGP was positively associated with the peripheral arterial calcification score (independently of age, gender, previous cardiovascular disease and t-ucMGP levels). CONCLUSIONS: High dp-ucMGP levels were independently associated with below-knee arterial calcification score in patients with type 2 diabetes and normal or slightly altered kidney function. The reversibility of the elevation of dp-ucMGP levels and the latter's relationship with clinical events merit further investigation.

Oxidised plant sterols as well as oxycholesterol increase the proportion of severe atherosclerotic lesions in female LDL receptor+/ - mice.

Oxysterols (oxidised cholesterol) may play a role in the pathogenesis of CVD. Similar to cholesterol, plant sterols are susceptible to oxidation. However, less is known about the potential atherogenicity of oxidised plant sterols (oxyphytosterols). In the present study, the atherogenicity of a mixture of oxyphytosterols was examined by feeding female LDL receptor-deficient (LDLR+/ -) mice for 35 weeks a control diet (atherogenic high-fat diet; n 9), an oxysterol diet (control diet+0·025 % (w/w) oxysterols; n 12) or an oxyphytosterol diet (control diet+0·025 % (w/w) oxyphytosterols; n 12). In the LDLR+/ - mice, serum levels of cholesterol, lipoprotein profiles, cholesterol exposure and inflammatory markers at the end of the experiment were comparable between the three diet groups. Nevertheless, the proportion of severe atherosclerotic lesions was significantly higher after oxysterol (41 %; P= 0·004) and oxyphytosterol (34 %; P= 0·011) diet consumption than after control diet consumption (26 %). Oxyphytosterol levels in the lesions were the highest in the oxyphytosterol group. Here, we show that not only dietary oxysterols but also dietary oxyphytosterols increase the proportion of severe atherosclerotic lesions. This suggests that plant sterols when oxidised may increase atherosclerotic lesion severity instead of lowering the size and severity of lesions when fed in their non-oxidised form. Therefore, this finding might give an indication as to where to find the answer in the current hot debate about the potential atherogenicity of plant sterols. However, to what extent these results can be extrapolated to the human situation warrants further investigation.

The role of vitamin K in soft-tissue calcification.

Seventeen vitamin K-dependent proteins have been identified to date of which several are involved in regulating soft-tissue calcification. Osteocalcin, matrix Gla protein (MGP), and possibly Gla-rich protein are all inhibitors of soft-tissue calcification and need vitamin K-dependent carboxylation for activity. A common characteristic is their low molecular weight, and it has been postulated that their small size is essential for calcification inhibition within tissues. MGP is synthesized by vascular smooth muscle cells and is the most important inhibitor of arterial mineralization currently known. Remarkably, the extrahepatic Gla proteins mentioned are only partly carboxylated in the healthy adult population, suggesting vitamin K insufficiency. Because carboxylation of the most essential Gla proteins is localized in the liver and that of the less essential Gla proteins in the extrahepatic tissues, a transport system has evolved ensuring preferential distribution of dietary vitamin K to the liver when vitamin K is limiting. This is why the first signs of vitamin K insufficiency are seen as undercarboxylation of the extrahepatic Gla proteins. New conformation-specific assays for circulating uncarboxylated MGP were developed; an assay for desphospho-uncarboxylated matrix Gla protein and another assay for total uncarboxylated matrix Gla protein. Circulating desphospho-uncarboxylated matrix Gla protein was found to be predictive of cardiovascular risk and mortality, whereas circulating total uncarboxylated matrix Gla protein was associated with the extent of prevalent arterial calcification. Vitamin K intervention studies have shown that MGP carboxylation can be increased dose dependently, but thus far only 1 study with clinical endpoints has been completed. This study showed maintenance of vascular elasticity during a 3-y supplementation period, with a parallel 12% loss of elasticity in the placebo group. More studies, both in healthy subjects and in patients at risk of vascular calcification, are required before conclusions can be drawn.

Low-dose menaquinone-7 supplementation improved extra-hepatic vitamin K status, but had no effect on thrombin generation in healthy subjects.

Vitamin K is required for the carboxylation of Gla-proteins in the liver (coagulation factors) and extra-hepatic tissues, such as bone (osteocalcin, OC), and arterial wall (matrix Gla-protein, MGP). Although the coagulation factors are essentially fully carboxylated under normal conditions, 10-40 % of OC and MGP remains undercarboxylated. We were therefore interested to study the dose-response effects of extra intake of menaquinones on the carboxylation of the extra-hepatic Gla-proteins. A total of forty-two healthy Dutch men and women aged between 18 and 45 years were randomised into seven groups to receive: placebo capsules or menaquinone-7 (MK-7) capsules at a daily dose of 10, 20, 45, 90, 180 or 360 µg. Circulating uncarboxylated OC (ucOC), carboxylated OC (cOC) and desphospho-uncarboxylated MGP were measured by ELISA. The ucOC:cOC ratio was calculated from circulating ucOC and cOC values. Endogenous thrombin potential and peak height were determined by calibrated automated thrombography. To increase the statistical power, we collapsed the treatment groups into three dosage groups: placebo, low-dose supplementation (doses below RDA, Commission Directive 2008/100/EC), and high-dose supplementation (doses around RDA, Commission Directive 2008/100/EC). MK-7 supplementation at doses in the order of the RDA (Commission Directive 2008/100/EC) increased the carboxylation of circulating OC and MGP. No adverse effects on thrombin generation were observed. Extra MK-7 intake at nutritional doses around the RDA (Commission Directive 2008/100/EC) improved the carboxylation of the extra-hepatic vitamin K-dependent proteins. Whether this improvement contributes to public health, i.e. increasing the protection against age-related diseases needs further investigation in specifically designed intervention trials.

Association of vitamin K status with adiponectin and body composition in healthy subjects: uncarboxylated osteocalcin is not associated with fat mass and body weight.

Osteocalcin (OC) is a vitamin K-dependent protein found in bone and in circulation. High serum γ-carboxylated OC reflects a high, and high uncarboxylated OC (ucOC) reflects a low vitamin K status. A revolutionary hypothesis is that ucOC acts as a hormone improving glucose handling and reducing fat mass. The objective was to test the logical extrapolation of the ucOC hormone hypothesis to humans that elevated ucOC is associated with higher body weight, BMI and fat mass. In a cross-sectional analysis, the associations of vitamin K status with circulating adiponectin and body composition were investigated in 244 postmenopausal women (study I). The effects of vitamin K treatment on adiponectin, body weight and BMI were investigated in archived samples from forty-two young men and women who received varying doses of menaquinone-7 during 12 weeks (study II) and from a cohort of 164 postmenopausal women who participated in a 3-year placebo-controlled trial on 45 mg menaquinone-4 (MK-4) (study III). No association was found between vitamin K status and circulating adiponectin before or after vitamin K supplementation. A higher carboxylation of OC was significantly correlated with lower body weight, BMI and fat mass of the trunk. Women taking MK-4 maintained their baseline body weight and BMI, whereas women taking placebo showed significant increases in both indices. These findings demonstrate that a high vitamin K status of bone has no effect on circulating adiponectin in healthy people and long-term vitamin K supplementation does not increase weight in healthy postmenopausal women.

Vitamin K, osteoporosis and degenerative diseases of ageing.

The function of vitamin K is to serve as a co-factor during the post-translational carboxylation of glutamate (Glu) residues into γ-carboxyglutamate (Gla) residues. The vital importance of the Gla-proteins essential for normal haemostasis is well recognized. During recent years, new Gla-containing proteins have been discovered and the vitamin K-dependent carboxylation is also essential for their function. It seems, however, that our dietary vitamin K intake is too low to support the carboxylation of at least some of these Gla-proteins. According to the triage theory, long-term vitamin K inadequacy is an independent, but modifiable risk factor for the development of degenerative diseases of ageing including osteoporosis and atherosclerosis.

Plant stanol supplementation decreases serum triacylglycerols in subjects with overt hypertriglyceridemia.

Evidence is accumulating that high serum concentrations of triacylglycerols (TAG) are, like LDL cholesterol, causally related to cardiovascular disease. A recent meta-analysis has indicated that plant stanol ester (PSE) intake not only lowered LDL cholesterol, but also serum TAG concentrations, especially in subjects with high baseline TAG concentrations. We therefore evaluated the effects of PSE supplementation on lipid metabolism in a population with elevated fasting TAG concentrations. In a randomized, placebo-controlled, parallel study, 28 subjects with elevated TAG concentrations (>1.7 mmol/L) were studied. After a 1-week run-in period during which a control margarine was used, subjects consumed for 3 weeks either control or PSE-enriched margarine (2.5 g/day of plant stanols). Serum plant stanol concentrations increased in all subjects receiving the PSE-enriched margarines, demonstrating good compliance. PSE supplementation significantly decreased serum total (6.7%, P = 0.015) and LDL cholesterol (9.5%, P = 0.041). A significant interaction between baseline TAG concentrations and PSE intake was found; PSE intake lowered TAG concentrations, particularly in subjects with high baseline TAG concentrations (>2.3 mmol/L; P = 0.009). Additionally, a significant interaction between baseline total number of LDL particles (LDL-P) and PSE intake was found (P = 0.020). PSE consumption lowered LDL-P, primarily in subjects with elevated baseline values; this was mainly due to a non-significant decrease in the number of atherogenic small LDL-P. Circulating levels of hs-CRP, glucose, and insulin were not changed after PSE intake. Taken together, PSE supplementation not only lowered LDL cholesterol, but also serum TAG concentrations, especially in subjects with overt hypertriglyceridemia.

Consumption of oat beta-glucan with or without plant stanols did not influence inflammatory markers in hypercholesterolemic subjects.

We have earlier demonstrated that muesli enriched with oat beta-glucan effectively lowered serum LDL cholesterol. Addition of plant stanols further lowered LDL cholesterol. Besides these hypocholesterolemic effects, beta-glucan and plant stanol esters (PSE) may also affect inflammatory processes. Forty-two mildly hypercholesterolemic subjects randomly consumed for 4 wk (crossover design) control muesli (4.8 g control fiber), beta-glucan muesli (4.8 g oat beta-glucan), or combination muesli (4.8 g oat beta-glucan plus 1.4 g stanol as PSE). Changes in cytokine production (IL-6, IL-8, and TNF-alpha) of LPS-stimulated peripheral blood mononuclear cells (PBMC) and whole blood were evaluated, as well as changes in plasma high-sensitivity (hs)-CRP. Additionally, changes in expression profiles of 84 genes involved in atherosclerosis metabolism were assessed in isolated PBMC. IL-6, IL-8, and TNF-alpha production by PBMC and whole blood after LPS stimulation did not differ between the treatments. Also high-sensitivity C-reactive protein (hs-CRP) levels were similar. beta-Glucan consumption did not change gene expression, while only 3 genes (ADFP, CDH5, CSF2) out of the 84 genes from the atherosclerotic risk panel were differentially expressed (p < 0.05) after consumption of PSE. Consumption of beta-glucan with or without PSE did not influence inflammatory parameters in mildly hypercholesterolemic subjects.

Water-soluble dietary fibers and cardiovascular disease.

One well-established way to reduce the risk of developing cardiovascular disease (CVD) is to lower serum LDL cholesterol levels by reducing saturated fat intake. However, the importance of other dietary approaches, such as increasing the intake of water-soluble dietary fibers is increasingly recognized. Well-controlled intervention studies have now shown that four major water-soluble fiber types-beta-glucan, psyllium, pectin and guar gum-effectively lower serum LDL cholesterol concentrations, without affecting HDL cholesterol or triacylglycerol concentrations. It is estimated that for each additional gram of water-soluble fiber in the diet serum total and LDL cholesterol concentrations decrease by -0.028 mmol/L and -0.029 mmol/L, respectively. Despite large differences in molecular structure, no major differences existed between the different types of water-soluble fiber, suggesting a common underlying mechanism. In this respect, it is most likely that water-soluble fibers lower the (re)absorption of in particular bile acids. As a result hepatic conversion of cholesterol into bile acids increases, which will ultimately lead to increased LDL uptake by the liver. Additionally, epidemiological studies suggest that a diet high in water-soluble fiber is inversely associated with the risk of CVD. These findings underlie current dietary recommendations to increase water-soluble fiber intake.

Simultaneous intake of beta-glucan and plant stanol esters affects lipid metabolism in slightly hypercholesterolemic subjects.

Intake of food products rich in water-soluble fiber beta-glucan and products enriched with plant stanol esters lower serum cholesterol. Combining 2 functional food ingredients into one food product may achieve additional reductions of serum cholesterol. Our objective was to investigate the effects of a simultaneous intake of beta-glucan plus plant stanol esters on lipid metabolism in mildly hypercholesterolemic volunteers. In a randomized, controlled, 3-period crossover study, 40 mildly hypercholesterolemic men and women received muesli in random order twice a day for 4 wk, which provided, in total, 5 g control fiber from wheat (control muesli), 5 g oat beta-glucan (beta-glucan muesli), or 5 g oat beta-glucan plus 1.5 g plant stanols (combination muesli). beta-Glucan muesli decreased serum LDL cholesterol by 5.0% compared with control muesli (P = 0.013). Combination muesli reduced LDL cholesterol by 9.6% compared with control muesli (P < 0.001), and by 4.4% compared with beta-glucan muesli (P = 0.036). Serum HDL cholesterol and triacylglycerol concentrations did not differ after the 3 treatments. Compared with control muesli, beta-glucan muesli increased bile acid synthesis (P = 0.043) and decreased cholesterol absorption (P = 0.011). Addition of plant stanols did not influence bile acid synthesis but decreased cholesterol absorption (P < 0.001) and raised cholesterol synthesis (P = 0.016) compared with control muesli, and the plant stanols decreased cholesterol absorption compared with beta-glucan muesli (P = 0.004). The combination muesli decreased serum concentrations of sitostanol compared with control muesli (P = 0.010). Plasma concentrations of lipid-soluble antioxidants did not differ after the 3 treatments. beta-Glucan muesli effectively lowered serum LDL cholesterol concentrations. The addition of plant stanol esters to beta-glucan-enriched muesli further lowered serum LDL cholesterol, although effects were slightly less than predicted.