Background. Vascular calcification is highly prevalent in persons with chronic kidney disease (CKD) and predicts cardiovascular disease (CVD) events. Matrix Gla protein (MGP) is a potent inhibitor of vascular calcification, and lower levels of its precursor—uncarboxylated MGP (ucMGP)—are associated with vascular calcification and atherosclerosis. Whether mild to moderate decrements in kidney function are associated with lower serum ucMGP is unknown.
Methods. In a cross-sectional study among 842 outpatients with stable CVD, estimated glomerular filtration rate (eGFR), serum cystatin-C and urine albumin-to-creatinine ratio (ACR) were measured and serum ucMGP levels were determined by ELISA. Multivariate linear regression evaluated the association of each kidney function measure with serum ucMGP levels.
Results. The mean eGFR was 76 ± 23 mL/min/1.73 m2, and 186 subjects (22%) had moderate CKD (eGFR <60 mL/min/1.73 m2). The mean ± SD ucMGP level was 3289 ± 1177 nM. In unadjusted analysis, each 10 mL/ min/1.73 m2 lower eGFR was associated with 101 nM lower ucMGP level. This association was only minimally attenuated in final multivariate models wherein each 10 mL/ min/1.73 m2 lower eGFR was associated with 79 nM lower ucMGP (95% confidence interval [CI]; 44 to 115; P < 0.001) after adjustment for age, sex, race, body mass index, blood pressure, smoking, hypertension, diabetes; and serum albumin, calcium, phosphorus, and fetuin-A levels. Similarly, in models adjusted for identical covariates, each 0.1 mg/L higher cystatin-C was associated with 39 nM lower ucMGP (95% CI 23 to 55; P < 0.001). In contrast, no significant association was observed between ACR and ucMGP in either unadjusted or adjusted analyses (adjusted P = 0.17). All associations were similar among subjects with or without diabetes (P-values for interaction > 0.50).
Conclusions. Among outpatients with stable CVD, a reduced glomerular filtration rate is associated with a decreased serum ucMGP level. In contrast, ACR is not associated with ucMGP levels. Whether ucMGP is a useful marker of vascular calcification and CVD event risk in persons with CKD deserves future study.
atherosclerosis; chronic kidney disease; matrix Gla protein; vascular calcification
Vascular calcification occurs when calcium accumulates in the intima (associated with atherosclerosis) and/or media layers of the vessel wall. Coronary artery calcification (CAC) reflects the calcium burden within the intima and media of the coronary arteries. In population-based studies, CAC independently predicts cardiovascular disease (CVD) and mortality. A preventive role for vitamin K in vascular calcification has been proposed based on its role in activating matrix Gla protein (MGP), a calcification inhibitor that is expressed in vascular tissue. Although animal and in vitro data support this role of vitamin K, overall data from human studies are inconsistent. The majority of population-based studies have relied on vitamin K intake to measure status. Phylloquinone is the primary dietary form of vitamin K and available supplementation trials, albeit limited, suggest phylloquinone supplementation is relevant to CAC. Yet observational studies have found higher dietary menaquinone, but not phylloquinone, to be associated with less calcification. Vascular calcification is highly prevalent in certain patient populations, especially in those with chronic kidney disease (CKD), and it is plausible vitamin K may contribute to reducing vascular calcification in patients at higher risk. Subclinical vitamin K deficiency has been reported in CKD patients, but studies linking vitamin K status to calcification outcomes in CKD are needed to clarify whether or not improving vitamin K status is associated with improved vascular health in CKD. This review summarizes the available evidence of vitamin K and vascular calcification in population-based studies and clinic-based studies, with a specific focus on CKD patients.
Mitral annular calcification (MAC) and aortic stenosis (AS) are associated with systemic calcification and cardiovascular disease (CVD) events. Matrix Gla protein (MGP) is an inhibitor of vascular calcification and lower levels of its precursor – uncarboxylated MGP (ucMGP) – are associated with vascular calcification in pilot studies.
Methods and Results
In this cross-sectional study of 839 outpatients with stable CVD, we measured serum ucMGP, and evaluated MAC and AS by echocardiography. The association of ucMGP with MAC differed by diabetes status (interaction P<0.001). Among participants without diabetes (n=615), higher ucMGP (per standard deviation [1,178 nM] increase) was associated with lower odds of MAC (odds ratio [OR] 0.73; 95% confidence interval [CI] 0.55-0.97) in models adjusted for traditional CVD risk factors, C-reactive protein, and kidney function. Among persons with diabetes (n=221), higher ucMGP was associated with higher odds of MAC (OR 1.89; 95% CI 1.29-2.78). Results were qualitatively similar for the association of ucMGP with AS although not statistically significant.
Among outpatients with stable CVD, higher ucMGP is associated with lower odds of MAC in persons without diabetes, and higher odds of MAC in persons with diabetes. Future studies should determine whether ucMGP levels are associated with CVD events, and whether such associations differ by diabetes status.
aortic stenosis; calcification; diabetes mellitus; matrix Gla protein; mitral annular 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.
Fibroblast growth factor 23 (FGF23), uncarboxylated matrix Gla protein (ucMGP), and fetuin-A are regulators of mineral metabolism and inhibitors of vascular calcification. Whether circulating levels of each are associated with cardiovascular disease (CVD) events or mortality in populations without end-stage renal disease is unknown.
To evaluate the associations of FGF23, ucMGP, and fetuin-A with mortality and CVD events.
12 outpatient clinics in the San Francisco Bay area.
833 outpatients with stable coronary artery disease (CAD), recruited from 11 September 2000 to 20 December 2002.
Fibroblast growth factor 23, ucMGP, and fetuin-A concentrations were measured at baseline. Participants were followed until 1 December 2008 for mortality and CVD events.
During a median follow-up of 6.0 years, 220 participants died and 182 had CVD events. Compared with participants with FGF-23 levels in the lowest tertile, those in the highest tertile had 2-fold greater risk for mortality (hazard ratio [HR], 2.15 [95% CI, 1.43 to 3.24]) and CVD events (HR, 1.83 [CI, 1.15 to 2.91]) after adjustment for traditional CVD risk factors, C-reactive protein levels, and kidney function. The highest ucMGP tertile was associated with lower mortality risk (HR, 0.48 [CI, 0.31 to 0.75]) and showed a nonsignificant trend toward lower CVD event risk by tertile analysis (HR, 0.65 [CI, 0.40 to 1.05])—an association that was significant when modeled continuously (P = 0.029). No significant association of fetuin-A with mortality (HR, 0.84 [CI, 0.55 to 1.27]) or CVD events (HR, 0.99 [CI, 0.64 to 1.55]) was observed.
Participants had prevalent CAD.
In outpatients with stable CAD, higher FGF23 and lower ucMGP levels are independently associated with mortality and CVD events.
Primary Funding Source
American Heart Association.
The human trabecular meshwork (TM) expresses many genes that have been associated with physiological (bone, cartilage, teeth) and pathological (vascular systems, kidney) calcification. In particular, the TM highly expresses the inhibitor of calcification Matrix Gla (MGP) gene, which encodes a vitamin K-dependent protein that requires post-translational activation to inhibit the formation of calcium precipitates. TM cells have high activity of the activating γ-carboxylase enzyme and produce active MGP. Silencing MGP increases the activity of alkaline phosphatase (ALP), an enzyme of the matrix vesicles and marker of calcification. Overexpressing MGP reduces the ALP activity induced by bone morphogenetic 2 (BMP2), a potent inducer of calcification. In this review we gathered evidence for the existence of a mineralization process in the TM. We selected twenty regulatory calcification genes, reviewed their functions in their original tissues and looked at their relative abundance in the TM by heat maps derived from existing microarrays. Although results are not yet fully conclusive and more experiments are needed, examining TM expression in the light of the calcification literature brings up many similarities. One such parallel is the role of mechanical forces in bone induction and the high levels of mineralization inhibitors found in the constantly mechanically stressed TM. During the next few years, examination of other calcification-related regulatory genes and pathways, as well as morphological examination of knockout animals would help elucidate the relevance of a calcification process to TM overall function.
Human trabecular meshwork; Perfused anterior segments, primary HTM cells, Calcification genes; Microarrays; Heat maps
Vitamin K is essential for the synthesis of proteins belonging to the Gla-protein family. To the members of this family belong four blood coagulation factors, which all are exclusively formed in the liver. The importance of vitamin K for hemostasis is demonstrated from the fact that vitamin K-deficiency is an acute, life-threatening condition due to excessive bleeding. Other members of the Gla-protein family are osteocalcin, matrix Gla-protein (MGP), and Gas6 that play key functions in maintaining bone strength, arterial calcification inhibition, and cell growth regulation, respectively. In total 17 Gla-proteins have been discovered at this time. Recently, it was observed that the dietary vitamin K requirement for the synthesis of the coagulation factors is much lower than for that of the extra-hepatic Gla-proteins. This forms the basis of the triage theory stating that during poor dietary supply, vitamins are preferentially utilized for functions that are important for immediate survival. This explains why in the healthy population all clotting factors are synthesized in their active form, whereas the synthesis of other Gla-proteins is sub-optimal in non-supplemented subjects. Prolonged sub-clinical vitamin K deficiency is a risk factor for osteoporosis, atherosclerosis, and cancer. Present recommendations for dietary intake are based on the daily dose required to prevent bleeding. Accumulating scientific data suggests that new, higher recommendations for vitamin K intake should be formulated.
phylloquinone; menaquinone; cardiovascular disease; osteoporosis; triage
Vitamin D deficiency is common in dialysis patients, whereas vitamin K status is less investigated despite its important implications for bone metabolism (bone Gla protein is vitamin K-dependent) and for vascular calcifications (matrix Gla protein is vitamin K-dependent).
Materials and methods:
The aim of the study was to assess the prevalence of vitamin D and K deficiency and the presence of vertebral fractures and vascular calcifications in haemodialysis patients (compared with a healthy control group). Subjects: 68 patients, 49 males and 19 females, mean age 66.62 years (± SD 11.3), undergoing thrice-weekly haemodialysis; mean dialytic age: 68.14±56.14 months.
The presence of vertebral fractures was assessed by means of vertebral morphometry (D5–L4) using a quantitative, computerised method (MorphoXpress).
The presence of vascular calcifications was assessed by means of vertebral spinal X-ray in L-L.
We measured biohumoural bone-vascular mineral metabolism parameters: total BGP and decarboxylated BGP (ucBGP), total MGP and decarboxylated MGP (ucMGP).
The presence of vertebral fractures was taken to correspond to a >20% reduction in the height of the vertebral body; a reduction of between 15 and 20% was considered borderline (B).
In the patients, versus controls, there emerged: deficit of 25(OH)D (98%, 60% carenti-38% insufficienti); vitamin K1 deficiency 32.08%; increased total BGP and ucBGP, increase in total MGP and reduction of ucMGP.
The prevalence of vertebral fractures was 57.35%+B: 27.94%. Vertebral fractures were associated with: anagraphical age (p=0.028), P (p=0.0445) and total BGP (p=0.0420).
The prevalence of vascular calcifications was 84%. Vascular calcifications were associated with: anagraphical age (p=0.0205), Ca (p=0.0192) and ucMGP (0.0453).
Marked vitamin D and K deficiency was associated with a high prevalence of vertebral fractures and vascular calcifications in haemodialysis patients with biohumoural bone mineral metabolism parameters within the KDOQI targets. Vitamin K is an important new biomarker of the bone-vascular axis in patients with chronic renal insufficiency.
Matrix GLA protein (MGP), a γ-carboxyglutamic acid (GLA)–rich, vitamin K–dependent and apatite-binding protein, is a regulator of hypertrophic cartilage mineralization during development. However, MGP is produced by both hypertrophic and immature chondrocytes, suggesting that MGP's role in mineralization is cell stage–dependent, and that MGP may have other roles in immature cells. It is also unclear whether MGP regulates the quantity of mineral or mineral nature and quality as well. To address these issues, we determined the effects of manipulations of MGP synthesis and expression in (a) immature and hypertrophic chondrocyte cultures and (b) the chick limb bud in vivo. The two chondrocyte cultures displayed comparable levels of MGP gene expression. Yet, treatment with warfarin, a γ-carboxylase inhibitor and vitamin K antagonist, triggered mineralization in hypertrophic but not immature cultures. Warfarin effects on mineralization were highly selective, were accompanied by no appreciable changes in MGP expression, alkaline phosphatase activity, or cell number, and were counteracted by vitamin K cotreatment. Scanning electron microscopy, x-ray microanalysis, and Fourier-transform infrared spectroscopy revealed that mineral forming in control and warfarin-treated hypertrophic cell cultures was similar and represented stoichiometric apatite. Virally driven MGP overexpression in cultured chondrocytes greatly decreased mineralization. Surprisingly, MGP overexpression in the developing limb not only inhibited cartilage mineralization, but also delayed chondrocyte maturation and blocked endochondral ossification and formation of a diaphyseal intramembranous bone collar. The results show that MGP is a powerful but developmentally regulated inhibitor of cartilage mineralization, controls mineral quantity but not type, and appears to have a previously unsuspected role in regulating chondrocyte maturation and ossification processes.
chondrocytes; matrix GLA protein; mineralization; ossification; limb development
The transformation of smooth muscle cells (VSMCs) in the vessel wall to osteoblast like cells is known to precede arterial calcification which may cause bleeding complications. The vitamin K-dependent protein MGP has been identified as an inhibitor of this process by binding BMP-2, a growth factor known to trigger the transformation. In this study, we determined if the vitamin K-dependent Gla region in MGP by itself can inhibit the growth factor activity of BMP-2 and if menaquinone-4 (MK4) regulates gene expression in VSMCs.
Materials and Methods
A synthetic γ-carboxyglutamic acid (Gla) containing peptide covering the Gla region in human MGP was used to test its ability to inhibit BMP-2 induced transformation of mouse pro-myoblast C2C12 cells into osteoblasts. MK4 was tested by microarray analysis as a gene regulatory molecule in VSMCs.
Results and Conclusions
The results show that the Gla- but not the Glu-peptide inhibited the transformation which provide evidence that the Gla region in MGP is directly involved in the BMP-2/MGP interaction and emphasizes the importance of the vitamin K-dependent modification of MGP. From the data obtained from the microarray analysis, we focused on two quantitatively altered cDNAs representing proteins known to be associated with vessel wall calcification. DT-diaphorase of the vitamin K-cycle, showed increased gene expression with a 4.8-fold higher specific activity in MK4 treated cells. Osteoprotegrin gene expression was down regulated and osteoprotegrin protein secretion from the MK4 treated cells was lowered 1.8-fold. These findings suggest that MK4 acts as an anti-calcification component in the vessel wall.
Vitamin K; Arterial calcification; Matrix Gla Protein (MGP); MK4 is an abbreviation for vitamin K4 of the vitamin K2 compounds
The pathogenesis of valvar calcification, which complicates the course of cardiac valve disease and also affects tissue valve prostheses, is incompletely understood. The present work explores the possible role of the vitamin K-dependent, calcium-binding amino acid, γ-carboxyglutamic acid (Gla) in valve mineralization. Gla is normally found in the vitamin K-dependent clotting factor proteins, and is also present in unique calcium binding proteins in bone, kidney, and lung. Unique Gla-containing proteins have also been isolated from pathologic calcifications including calcium containing renal stones and calcified atherosclerotic plaque. Calcified valves including specimens with calcific aortic stenosis, calcified porcine xenograft valves, and a calcified aortic homograft valve were analyzed for Gla content, complete amino acid analysis, and tissue calcium and phosphorus levels. Normal porcine valves contained protein-bound Gla (2.0-10.6 Gla/104 amino acids): no Gla was present in normal valve leaflets. Furthermore, Gla levels paralleled tissue calcium content in the calcified valves. In addition, complete amino acid analysis indicated a decline in valvar collagen content plus increased acidic proteins in conjunction with valvar calcification and the presence of Gla-containing proteins. These results suggest that calcific valvar disease may result in part from vitamin K-dependent processes.
Pseudoxanthoma elasticum (PXE) is a heritable disease characterized by calcified elastic fibers in cutaneous, ocular and vascular tissues. PXE is caused by mutations in ABCC6, which encodes a protein of the ATP-driven organic anion transporter family. The inability of this transporter to secrete its substrate into the circulation is the likely cause of PXE. Vitamin K plays a role in the regulation of mineralization processes as a co-factor in the carboxylation of calcification inhibitors such as Matrix Gla Protein (MGP). Vitamin K precursor or a conjugated form has been proposed as potential substrate(s) for ABCC6. We investigated whether an enriched diet of vitamin K1 or vitamin K2 (MK4) could stop or slow the disease progression in Abcc6-/- mice. Abcc6-/- mice were placed on a diet of either vitamin K1 or MK4 at 5 or 100 mg/kg at prenatal, 3 weeks or 3 months of age. Disease progression was quantified by measuring the calcium content of one side of the mouse muzzle skin and histological staining for calcium of the opposing side. Raising the vitamin K1 or MK4 content of the diet increased the concentration of circulating MK4 in the serum. However, this increase did not significantly affect the MGP carboxylation status or reduce its abnormal abundance, the total calcium content or the pathologic calcification in the whiskers of the 3 treatment groups compared to controls. Our findings showed that raising the dietary intake of vitamin K1 or MK4 was not beneficial in the treatment of PXE and suggested that the availability of vitamin K may not be a limiting factor in this pathology.
pseudoxanthoma elasticum; vitamin K; mineralization; Abcc6; mouse
Factors associated with mineralization and osteophyte formation in osteoarthritis (OA) are incompletely understood. Genetic polymorphisms of matrix Gla protein (MGP), a mineralization inhibitor, have been associated clinically with conditions of abnormal calcification. We therefore evaluated the relationship of MGP concentrations and polymorphisms at the MGP locus to hand OA.
Ours was an ancillary study to a 3-year randomized trial assessing the effect of vitamin K supplementation on vascular calcification and bone loss among community-dwelling elders. We studied participants who had serum MGP concentration measured and DNA genotyped for 3 MGP genetic polymorphisms (rs1800802, rs1800801, and rs4236), and who had hand radiographs. We evaluated the cross-sectional associations of serum MGP and the 3 MGP genetic polymorphisms, respectively, with radiographic hand OA using logistic regression with generalized estimating equations, adjusting for potential confounders.
Radiographic hand OA in ≥ 1 joint was present in 71% of the 376 participants (mean age 74 years, mean body mass index 28 kg/m2, 59% women). No significant association between serum MGP concentrations and radiographic hand OA was found [adjusted OR 1.0 (ref), 1.40, 1.21, and 1.21 for quartiles 1–4, respectively]. Homozygosity of the rs1800802 minor allele was associated with 0.56 times lower prevalence of hand OA compared with having ≥ 1 major allele at this locus (95% CI 0.32–0.99, p = 0.046).
There may be an association between hand OA and genetic polymorphism at the MGP locus that is not reflected by total MGP serum concentrations. Further studies are warranted to replicate and elucidate potential mechanisms underlying these observed associations.
MATRIX GLA PROTEIN; POLYMORPHISM; OSTEOARTHRITIS; VITAMIN K
Plasma antioxidant vitamins (retinol, α-tocopherol, β-carotene) were measured to establish the influence of clinical condition and mycophenolate mofetil (MMF) treatment on the nutritional status of renal transplant recipients.
Material and methods
In 106 adult patients plasma vitamins were measured and 24-h diet history questionnaires were conducted. The MMF influence on plasma vitamins was verified in 61 patients.
The current dietary intakes of vitamins in daily food rations were lower than recommended. Plasma retinol was lower in patients suffering from gastrointestinal disorders (1.25 ±0.48 mg/l vs. 1.55 ±0.70 mg/l) and inversely associated with aminotransferases activity (p = 0.019) and creatinine clearance (p = 0.021). Retinol concentrations were positively associated with plasma creatinine (p = 0.027) and pharmacokinetic parameters of MMF phenyl glucuronide. β-Carotene concentrations were higher in women (0.39 ±0.46 mg/l vs. 0.28 ±0.23 mg/l; p = 0.041) and when MMF was co-administered with cyclosporine vs. tacrolimus (0.45 ±0.62 mg/l vs. 0.25 ±0.19 mg/l). Plasma α-tocopherol correlated negatively with the mycophenolic acid pre-dose concentration (p = 0.027) and was significantly lower in patients treated with calcineurin inhibitors (8.90 ±5.23 mg/l vs. 12.25 ±5.62 mg/l). A positive correlation was observed between α-tocopherol levels and aspartate aminotransferase (p = 0.006). In multivariate regression aspartate aminotransferase and MMF treatment significantly influenced retinol (p < 0.001).
The MMF treatment was associated with significantly lower retinol concentrations. The gastrointestinal disorders occurrence in MMF-treated patients may cause a decrease in retinol absorption. Diet adjustment and/or vitamin A supplementation should be considered.
antioxidant vitamins; clinical condition; mycophenolate mofetil; renal transplantation; nutritional status
Like vitamin D deficit, magnesium deficit is considered to be a risk factor for cardiovascular disease. Several steps in the vitamin D metabolism, such as vitamin D binding to its transport protein and the conversion of vitamin D into the hormonal form 1,25-dihydroxyvitamin D by hepatic and renal hydroxylation, depend on magnesium as a cofactor. A new analysis of two National Health and Nutrition Examination Surveys data sets, published in BMC Medicine, investigated potential interactions between magnesium intake, circulating 25-hydroxyvitamin D, which is the generally accepted indicator of vitamin D status, and mortality. Data indicate a reduced risk of insufficient/deficient vitamin D status at high magnesium intake and an inverse association between circulating 25-hydroxyvitamin D and mortality, particularly cardiovascular mortality, among those with magnesium intake above the median. The study provides important findings concerning potential metabolic interactions between magnesium and vitamin D and its clinical relevance. However, results should be considered preliminary since biochemical data on individual magnesium status were lacking, confounding cannot be excluded and questions on the dose?response relationship still remain to be answered.
Please see related research article: http://www.biomedcentral.com/1741-7015/11/187.
25-hydroxyvitamin D; Cardiovascular mortality; Magnesium; Mortality; Vitamin D deficit
Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. Smooth muscle cells (SMCs) may play an important role in vascular cartilaginous metaplasia and calcification via reprogramming to the osteochondrogenic state. To study whether SM lineage reprogramming and thus matrix calcification is reversible and what the necessary regulatory factors are to reverse this process, we used cells isolated from calcifying arterial medias of 4-week-old matrix Gla protein knockout mice (MGP−/− SMCs). We found that vascular cells with an osteochondrogenic phenotype regained SMC properties (positive for SM22α and SM α-actin) and down-regulated osteochondrogenic gene expression (Runx2/Cbfa1 and osteopontin) upon culture in medium that favors SMC differentiation. Over time, the MGP−/− SMCs no longer expressed osteochondrogenic proteins and became indistinguishable from wild-type SMCs. Moreover, phenotypic switch of the restored SMCs to the osteochondrogenic state was re-induced by the pro-calcific factor, inorganic phosphate. Finally, loss- and gain-of-function studies of myocardin, a SM-specific transcription co-activator, and Runx2/Cbfa1, an osteochondrogenic transcription factor, revealed that upregulation of Runx2/Cbfa1, but not loss of myocardin, played a critical role in phosphate-induced SMC lineage reprogramming and calcification. These results are the first to demonstrate reversibility of vascular SMCs to an osteochondrogenic state in response to local environmental cues, and that myocardin-enforced SMC lineage allocation was not sufficient to block vascular calcification. On the other hand, Runx2/Cbfa1 was found to be a decisive factor identified in the process.
Calcification; Lineage reprogramming; Myocardin; Runx2/Cbfa1; Smooth muscle cells
Vitamin D status influences skeletal health, the risk of falls and fractures, and muscle health, and it has been associated with inflammatory, infectious, cardiovascular and metabolic disorders in addition to some cancers. Prevailing intracellular infections such as tuberculosis are speculated to relate to vitamin D status. The vitamin D sources are dietary and dermal, the latter depending on UVB radiation exposure from the sun. Life in the Arctic influences vitamin D status because of dietary peculiarities, the polar night, waning of the ozone layer and maybe ethnic differences between Inuit and non-Inuit.
Objective and design
Data on vitamin D status as estimated by plasma 25OHD in Inuit and non-Inuit in Greenland are reviewed.
Decreasing intake of vitamin D-rich local food items associated with decreasing plasma 25OHD levels and insufficient vitamin D status is seen with low intake of traditional Inuit foods. Plasma 25OHD levels increase markedly during spring and summer in parallel with the high influx of sunlight while plasma 25OHD is not influenced by obesity in Greenland Inuit and no clear-cut association is seen between plasma 25OHD and the risk of tuberculosis.
The frequency of vitamin D deficiency in populations in Greenland rises with the dietary transition and diseases related to low vitamin D status should be monitored.
vitamin D; diet; lifestyle changes; UVB radiation; obesity; Inuit Eskimos; Arctic Greenland; review
Matrix γ-carboxy glutamic acid protein (MGP) is a 10-kDa secreted protein containing five residues of the vitamin K-dependent calcium binding amino acid γ-carboxyglutamic acid (Gla). This study was carried out to examine the effects of MGP gene promoter polymorphism (T-138C) on blood lead levels (BLL) and hematological parameters in 113 battery manufacturing unit workers occupationally exposed to lead and 102 controls. Genotypes for the MGP T-138C polymorphism were determined by PCR and restriction fragment length digestion. BLL were determined by Anode Stripping Voltammetry using ESA Model 3010B Lead analyzer. Complete blood picture (CBP) was analyzed using ADVIA Cell counter for each sample. The frequencies of MGP–TT, CT and CC genotypes in our population were 38.6%, 44.3%, and 17.2%, respectively. The frequencies for T and C alleles were 0.612 and 0.386, respectively. Although BLL did not differ significantly among genotypes; they were higher in workers with TT/CT genotype compared to CC genotype subjects (76–88 μg/dL vs 22–45 μg/dL, p > 0.05). About 29.2% of volunteers (n = 33) from the occupationally exposed group had hemoglobin levels below 10.0 gms/dl. There was no significant difference in total white cell count and platelet count between occupational and non-exposed groups. The possible role of SNPs in the promoter region of MGP gene with relation to lead toxicity was investigated for the first time in the Indian population; although significance could not be achieved in this study, further assessments over a larger population size may help in better understanding of the consequences of lead exposure.
Lead toxicity; Blood lead levels; MGP gene polymorphisms; PCR
The kidneys are essential for the metabolism of vitamin A (retinol) and its transport proteins retinol-binding protein 4 (RBP4) and transthyretin. Little is known about changes in serum concentration after living donor kidney transplantation (LDKT) as a consequence of unilateral nephrectomy; although an association of these parameters with the risk of cardiovascular diseases and insulin resistance has been suggested. Therefore we analyzed the concentration of retinol, RBP4, apoRBP4 and transthyretin in serum of 20 living-kidney donors and respective recipients at baseline as well as 6 weeks and 6 months after LDKT.
As a consequence of LDKT, the kidney function of recipients was improved while the kidney function of donors was moderately reduced within 6 weeks after LDKT. With regard to vitamin A metabolism, the recipients revealed higher levels of retinol, RBP4, transthyretin and apoRBP4 before LDKT in comparison to donors. After LDKT, the levels of all four parameters decreased in serum of the recipients, while retinol, RBP4 as well as apoRBP4 serum levels of donors increased and remained increased during the follow-up period of 6 months.
LDKT is generally regarded as beneficial for allograft recipients and not particularly detrimental for the donors. However, it could be demonstrated in this study that a moderate reduction of kidney function by unilateral nephrectomy, resulted in an imbalance of components of vitamin A metabolism with a significant increase of retinol and RBP4 and apoRBP4 concentration in serum of donors.
Donors; glomerular filtration rate; kidney transplantation; retinol; retinol-binding protein 4; transthyretin
Calcification of renal allografts is common in the first year after transplantation and is related to hyperparathyroidism. It is associated with an impaired long-term function of the graft (Am J Transplant 5∶1934-41, 2005). Aim of this study is to examine the role of the anti-calcifying/calcifying factors in the pathophysiology of renal allograft calcification.
We analyzed protocol allograft biopsies, blood and urine samples of 31 patients with and 27 patients without allograft calcification taken at 6 weeks, 3 and 6 months after transplantation. Patient demographical data, cold ischemia time, initial graft function and donor characteristics were comparable between the two groups. Biopsies were stained for osteopontin, fetuin, and matrix-gla-protein. Serum and urine electrolytes, matrix-gla-protein, fetuin, Vitamin D and intact parathyroid hormone in serum and osteopontin (OPN) in urine were examined.
Serum levels of fetuin and matrix-Gla protein as well as urinary levels of OPN showed specific time dependent changes (6 weeks vs. 3 months vs. 6 months; all p<0.0001). In patients with calcifications, urinary levels of OPN were decreased by 55% at 6 weeks and increased thereafter, showing 54% higher levels at 6 months compared to patients without calcification (6 weeks: p<0.01, 6 months: p<0.05). Local protein expression of fetuin-A, matrix-Gla protein and OPN in the graft was specifically increased around calcified plaques, without differences in the mRNA tissue expression.
Anticalcifying factors show significant changes in the early phase after renal transplantation, which may be important for the prevention of allograft calcification. The differences in OPN indicate an involvement of this factor in the regulation of calcification.
Implanted silicone medical prostheses induce a dynamic sequence of histologic events in adjacent tissue resulting in the formation of a fibrotic peri-prosthetic capsule. In some cases, capsular calcification occurs, requiring surgical intervention. In this study we investigated capsules from silicone gel-filled breast prostheses to test the hypothesis that this calcification might be regulated by the small vitamin K-dependent protein, matrix Gla protein (MGP), a potent inhibitor of arterial calcification, or by Fetuin-A, a hepatocyte-derived glycoprotein also implicated as a regulator of pathologic calcification. Immunolocalization studies of explanted capsular tissue, using conformation-specific antibodies, identified the mineralization-protective γ-carboxylated MGP isomer (cMGP) within cells of uncalcified capsules, whereas the non-functional undercarboxylated isomer (uMGP) was typically absent. Both were upregulated in calcific capsules and co-localized with mineral plaque and adjacent fibers. Synovial-like metaplasia was present in one uncalcified capsule in which MGP species were differentially localized within the pseudosynovium. Fetuin-A was localized to cells within uncalcified capsules and to mineral deposits within calcific capsules. The osteoinductive cytokine bone morphogenic protein-2 localized to collagen fibers in uncalcified capsules. These findings demonstrate that MGP, in its vitamin K-activated conformer, may represent a pharmacological target to sustain the health of the peri-prosthetic tissue which encapsulates silicone breast implants as well as other implanted silicone medical devices.
The uncarboxylated form (ucOC), but not the γ-carboxylated form (GlaOC), of the bone-derived protein osteocalcin stimulates insulin secretion and regulates energy metabolism in insulin target tissues. Glucagon-like peptide–1 (GLP-1) is an insulin secretagogue that is released from the gut in response to food intake. We have now found that Gprc6a, a putative ucOC receptor, is expressed in epithelial cells of the mouse small intestine as well as in STC-1 enteroendocrine cells. Secretion of GLP-1 by STC-1 cells was stimulated by ucOC but not by GlaOC. The serum GLP-1 concentration in mice was increased by intraperitoneal or oral administration of ucOC, whereas GlaOC was effective in this regard only after oral application. Serum insulin levels were also increased by ucOC, and this effect was potentiated by an inhibitor of dipeptidyl peptidase IV and blocked by a GLP-1 receptor antagonist. Intravenous injection of ucOC in mice increased the serum GLP-1 concentration, and also increased the serum level of insulin. Our results suggest that ucOC acts via Gprc6a to induce GLP-1 release from the gut, and that the stimulatory effect of ucOC on insulin secretion is largely mediated by GLP-1.
Atherosclerotic coronary artery calcification (CAC) is associated with increased coronary heart disease (CHD) risk. Matrix Gla protein (MGP) is an inhibitor of calcification in vivo. However, little is known regarding the distribution of circulating MGP and its associations with CHD risk factors or with CAC in humans.
Methods and Results
Serum MGP concentrations were determined in 2 independent populations of men and women free of clinically apparent cardiovascular disease: study A, n=316, mean age 58 years, and study B, n=452, mean age 68 years. CAC was determined by computed tomography. Mean MGP concentrations were 98.4 and 198 ng/mL in men, and 97.4 and 201 ng/mL in women, in study A and study B, respectively. In both cohorts, MGP levels were higher with increasing age. In age-adjusted analyses, there was an association of circulating MGP with increasing Framingham CHD risk score (in study A, P=0.003 in men and P=0.016 in women, respectively; in study B, a nonsignificant increase in men and P=0.05 in women, respectively). Significant associations of circulating MGP with high-density lipoprotein and other individual CHD risk factors were also noted in both cohorts. There were no consistent associations between MGP and CAC after adjustment for CHD risk score in the 2 cohorts.
MGP is associated with individual CHD risk factors and the Framingham CHD risk score in men and women free of clinically apparent CHD. The relation of MGP with CAC deserves further study in larger populations.
atherosclerosis; coronary artery calcification; coronary risk factors; matrix Gla protein
Vascular calcification is a major risk factor for cardiovascular morbidity and mortality. In order to develop appropriate prevention and/or therapeutic strategies for vascular calcification, it is important to understand the origins of the cells that participate in this process. In this report, we used the SM22-Cre recombinase and Rosa26-LacZ alleles to genetically trace cells derived from smooth muscle. We found that smooth muscle cells (SMCs) gave rise to osteochondrogenic precursor- and chondrocyte-like cells in calcified blood vessels of matrix Gla protein deficient (MGP−/−) mice. This lineage reprogramming of SMCs occurred prior to calcium deposition, and was associated with an early onset of Runx2/Cbfa1 expression and the down regulation of myocardin and Msx2. There was no change in the constitutive expression of Sox9 or BMP2. Osterix, Wnt3a and Wnt7a mRNAs were not detected in either calcified MGP−/− or non-calcified wild type (MGP+/+) vessels. Finally, mechanistic studies in vitro suggest that Erk signaling might be required for SMC transdifferentiation under calcifying conditions. These results provide strong support for the hypothesis that adult SMCs can transdifferentiate and that SMC transdifferentiation is an important process driving vascular calcification and the appearance of skeletal elements in calcified vascular lesions.
Genetic fate mapping; Lineage reprogramming; Runx2/Cbfa1; Smooth muscle cells; Vascular calcification
The bone morphogenetic proteins (BMP), a family of morphogens, have been implicated as mediators of calcification and inflammation in the vascular wall.
To investigate the effect of altered expression of Matrix Gla Protein (MGP), an inhibitor of BMP, on vascular disease.
Methods and Results
We used MGP transgenic or MGP deficient mice bred to apolipoprotein E mice, a model of atherosclerosis. MGP overexpression reduced vascular BMP activity, atherosclerotic lesion size, intimal and medial calcification, and inflammation. It also reduced expression of the activin-like kinase receptor (ALK)1 and the vascular endothelial growth factor (VEGF), part of a BMP-activated pathway that regulates angiogenesis and may enhance lesion formation and calcification. Conversely, MGP deficiency increased BMP activity, which may explain the diffuse calcification of vascular medial cells in MGP deficient aortas, and the increase in expression of ALK1 and VEGF. Unexpectedly, atherosclerotic lesion formation was decreased in MGP deficient mice, which may be explained by a dramatic reduction in expression of endothelial adhesion molecules limiting monocyte infiltration of the artery wall.
Our results indicate that BMP signaling is a key regulator of vascular disease, requiring careful control to maintain normal vascular homeostasis.
Bone morphogenetic protein; Matrix Gla protein; Atherosclerosis; Vascular calcification; Inflammation