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1.  Association of kidney function and uncarboxylated matrix Gla protein: Data from the Heart and Soul Study 
Nephrology Dialysis Transplantation  2009;24(7):2095-2101.
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.
doi:10.1093/ndt/gfp024
PMCID: PMC2721482  PMID: 19204017
atherosclerosis; chronic kidney disease; matrix Gla protein; vascular calcification
2.  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.
doi:10.1186/1475-2840-13-85
PMCID: PMC4017083  PMID: 24762216
Matrix gla protein; Type 2 diabetes; Peripheral calcification
3.  The Association of Uncarboxylated Matrix Gla Protein with Mitral Annular Calcification Differs by Diabetes Status: The Heart and Soul Study 
Atherosclerosis  2009;210(1):320-325.
Objective
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.
Conclusions
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.
doi:10.1016/j.atherosclerosis.2009.11.023
PMCID: PMC2862076  PMID: 20015492
aortic stenosis; calcification; diabetes mellitus; matrix Gla protein; mitral annular calcification
4.  The Associations of Fibroblast Growth Factor 23 and Uncarboxylated Matrix Gla Protein With Mortality in Coronary Artery Disease: The Heart and Soul Study 
Annals of internal medicine  2010;152(10):640-648.
Background
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.
Objective
To evaluate the associations of FGF23, ucMGP, and fetuin-A with mortality and CVD events.
Design
Observational study.
Setting
12 outpatient clinics in the San Francisco Bay area.
Patients
833 outpatients with stable coronary artery disease (CAD), recruited from 11 September 2000 to 20 December 2002.
Measurements
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.
Results
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.
Limitation
Participants had prevalent CAD.
Conclusion
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.
doi:10.1059/0003-4819-152-10-201005180-00004
PMCID: PMC3079370  PMID: 20479029
5.  The Role of Vitamin K in Soft-Tissue Calcification1 
Advances in Nutrition  2012;3(2):166-173.
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.
doi:10.3945/an.111.001628
PMCID: PMC3648717  PMID: 22516724
6.  P23 - Vitamin D and K Deficiency in Haemodialysis Patients with a High Prevalence of Vertebral Fractures and Vascular Calcifications: A Preliminary Study 
Introduction:
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).
Results:
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).
Conclusions:
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.
PMCID: PMC3213784
7.  Vitamin K Status and Vascular Calcification: Evidence from Observational and Clinical Studies12 
Advances in Nutrition  2012;3(2):158-165.
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.
doi:10.3945/an.111.001644
PMCID: PMC3648716  PMID: 22516723
8.  Matrix Gla Protein Is Associated With Risk Factors for Atherosclerosis but not With Coronary Artery Calcification 
Objectives
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.
Conclusions
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.
doi:10.1161/01.ATV.0000245793.83158.06
PMCID: PMC2562549  PMID: 16973975
atherosclerosis; coronary artery calcification; coronary risk factors; matrix Gla protein
9.  Matrix Gla Protein Is a Developmental Regulator of Chondrocyte Mineralization And, When Constitutively Expressed, Blocks Endochondral and Intramembranous Ossification in the Limb 
The Journal of Cell Biology  1999;147(5):1097-1108.
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.
PMCID: PMC2169349  PMID: 10579728
chondrocytes; matrix GLA protein; mineralization; ossification; limb development
10.  Clinical Utility of Vitamin D Testing 
Executive Summary
This report from the Medical Advisory Secretariat (MAS) was intended to evaluate the clinical utility of vitamin D testing in average risk Canadians and in those with kidney disease. As a separate analysis, this report also includes a systematic literature review of the prevalence of vitamin D deficiency in these two subgroups.
This evaluation did not set out to determine the serum vitamin D thresholds that might apply to non-bone health outcomes. For bone health outcomes, no high or moderate quality evidence could be found to support a target serum level above 50 nmol/L. Similarly, no high or moderate quality evidence could be found to support vitamin D’s effects in non-bone health outcomes, other than falls.
Vitamin D
Vitamin D is a lipid soluble vitamin that acts as a hormone. It stimulates intestinal calcium absorption and is important in maintaining adequate phosphate levels for bone mineralization, bone growth, and remodelling. It’s also believed to be involved in the regulation of cell growth proliferation and apoptosis (programmed cell death), as well as modulation of the immune system and other functions. Alone or in combination with calcium, Vitamin D has also been shown to reduce the risk of fractures in elderly men (≥ 65 years), postmenopausal women, and the risk of falls in community-dwelling seniors. However, in a comprehensive systematic review, inconsistent results were found concerning the effects of vitamin D in conditions such as cancer, all-cause mortality, and cardiovascular disease. In fact, no high or moderate quality evidence could be found concerning the effects of vitamin D in such non-bone health outcomes. Given the uncertainties surrounding the effects of vitamin D in non-bone health related outcomes, it was decided that this evaluation should focus on falls and the effects of vitamin D in bone health and exclusively within average-risk individuals and patients with kidney disease.
Synthesis of vitamin D occurs naturally in the skin through exposure to ultraviolet B (UVB) radiation from sunlight, but it can also be obtained from dietary sources including fortified foods, and supplements. Foods rich in vitamin D include fatty fish, egg yolks, fish liver oil, and some types of mushrooms. Since it is usually difficult to obtain sufficient vitamin D from non-fortified foods, either due to low content or infrequent use, most vitamin D is obtained from fortified foods, exposure to sunlight, and supplements.
Clinical Need: Condition and Target Population
Vitamin D deficiency may lead to rickets in infants and osteomalacia in adults. Factors believed to be associated with vitamin D deficiency include:
darker skin pigmentation,
winter season,
living at higher latitudes,
skin coverage,
kidney disease,
malabsorption syndromes such as Crohn’s disease, cystic fibrosis, and
genetic factors.
Patients with chronic kidney disease (CKD) are at a higher risk of vitamin D deficiency due to either renal losses or decreased synthesis of 1,25-dihydroxyvitamin D.
Health Canada currently recommends that, until the daily recommended intakes (DRI) for vitamin D are updated, Canada’s Food Guide (Eating Well with Canada’s Food Guide) should be followed with respect to vitamin D intake. Issued in 2007, the Guide recommends that Canadians consume two cups (500 ml) of fortified milk or fortified soy beverages daily in order to obtain a daily intake of 200 IU. In addition, men and women over the age of 50 should take 400 IU of vitamin D supplements daily. Additional recommendations were made for breastfed infants.
A Canadian survey evaluated the median vitamin D intake derived from diet alone (excluding supplements) among 35,000 Canadians, 10,900 of which were from Ontario. Among Ontarian males ages 9 and up, the median daily dietary vitamin D intake ranged between 196 IU and 272 IU per day. Among females, it varied from 152 IU to 196 IU per day. In boys and girls ages 1 to 3, the median daily dietary vitamin D intake was 248 IU, while among those 4 to 8 years it was 224 IU.
Vitamin D Testing
Two laboratory tests for vitamin D are available, 25-hydroxy vitamin D, referred to as 25(OH)D, and 1,25-dihydroxyvitamin D. Vitamin D status is assessed by measuring the serum 25(OH)D levels, which can be assayed using radioimmunoassays, competitive protein-binding assays (CPBA), high pressure liquid chromatography (HPLC), and liquid chromatography-tandem mass spectrometry (LC-MS/MS). These may yield different results with inter-assay variation reaching up to 25% (at lower serum levels) and intra-assay variation reaching 10%.
The optimal serum concentration of vitamin D has not been established and it may change across different stages of life. Similarly, there is currently no consensus on target serum vitamin D levels. There does, however, appear to be a consensus on the definition of vitamin D deficiency at 25(OH)D < 25 nmol/l, which is based on the risk of diseases such as rickets and osteomalacia. Higher target serum levels have also been proposed based on subclinical endpoints such as parathyroid hormone (PTH). Therefore, in this report, two conservative target serum levels have been adopted, 25 nmol/L (based on the risk of rickets and osteomalacia), and 40 to 50 nmol/L (based on vitamin D’s interaction with PTH).
Ontario Context
Volume & Cost
The volume of vitamin D tests done in Ontario has been increasing over the past 5 years with a steep increase of 169,000 tests in 2007 to more than 393,400 tests in 2008. The number of tests continues to rise with the projected number of tests for 2009 exceeding 731,000. According to the Ontario Schedule of Benefits, the billing cost of each test is $51.7 for 25(OH)D (L606, 100 LMS units, $0.517/unit) and $77.6 for 1,25-dihydroxyvitamin D (L605, 150 LMS units, $0.517/unit). Province wide, the total annual cost of vitamin D testing has increased from approximately $1.7M in 2004 to over $21.0M in 2008. The projected annual cost for 2009 is approximately $38.8M.
Evidence-Based Analysis
The objective of this report is to evaluate the clinical utility of vitamin D testing in the average risk population and in those with kidney disease. As a separate analysis, the report also sought to evaluate the prevalence of vitamin D deficiency in Canada. The specific research questions addressed were thus:
What is the clinical utility of vitamin D testing in the average risk population and in subjects with kidney disease?
What is the prevalence of vitamin D deficiency in the average risk population in Canada?
What is the prevalence of vitamin D deficiency in patients with kidney disease in Canada?
Clinical utility was defined as the ability to improve bone health outcomes with the focus on the average risk population (excluding those with osteoporosis) and patients with kidney disease.
Literature Search
A literature search was performed on July 17th, 2009 using OVID MEDLINE, MEDLINE In-Process and Other Non-Indexed Citations, EMBASE, the Cumulative Index to Nursing & Allied Health Literature (CINAHL), the Cochrane Library, and the International Agency for Health Technology Assessment (INAHTA) for studies published from January 1, 1998 until July 17th, 2009. Abstracts were reviewed by a single reviewer and, for those studies meeting the eligibility criteria, full-text articles were obtained. Reference lists were also examined for any additional relevant studies not identified through the search. Articles with unknown eligibility were reviewed with a second clinical epidemiologist, then a group of epidemiologists until consensus was established. The quality of evidence was assessed as high, moderate, low or very low according to GRADE methodology.
Observational studies that evaluated the prevalence of vitamin D deficiency in Canada in the population of interest were included based on the inclusion and exclusion criteria listed below. The baseline values were used in this report in the case of interventional studies that evaluated the effect of vitamin D intake on serum levels. Studies published in grey literature were included if no studies published in the peer-reviewed literature were identified for specific outcomes or subgroups.
Considering that vitamin D status may be affected by factors such as latitude, sun exposure, food fortification, among others, the search focused on prevalence studies published in Canada. In cases where no Canadian prevalence studies were identified, the decision was made to include studies from the United States, given the similar policies in vitamin D food fortification and recommended daily intake.
Inclusion Criteria
Studies published in English
Publications that reported the prevalence of vitamin D deficiency in Canada
Studies that included subjects from the general population or with kidney disease
Studies in children or adults
Studies published between January 1998 and July 17th 2009
Exclusion Criteria
Studies that included subjects defined according to a specific disease other than kidney disease
Letters, comments, and editorials
Studies that measured the serum vitamin D levels but did not report the percentage of subjects with serum levels below a given threshold
Outcomes of Interest
Prevalence of serum vitamin D less than 25 nmol/L
Prevalence of serum vitamin D less than 40 to 50 nmol/L
Serum 25-hydroxyvitamin D was the metabolite used to assess vitamin D status. Results from adult and children studies were reported separately. Subgroup analyses according to factors that affect serum vitamin D levels (e.g., seasonal effects, skin pigmentation, and vitamin D intake) were reported if enough information was provided in the studies
Quality of Evidence
The quality of the prevalence studies was based on the method of subject recruitment and sampling, possibility of selection bias, and generalizability to the source population. The overall quality of the trials was examined according to the GRADE Working Group criteria.
Summary of Findings
Fourteen prevalence studies examining Canadian adults and children met the eligibility criteria. With the exception of one longitudinal study, the studies had a cross-sectional design. Two studies were conducted among Canadian adults with renal disease but none studied Canadian children with renal disease (though three such US studies were included). No systematic reviews or health technology assessments that evaluated the prevalence of vitamin D deficiency in Canada were identified. Two studies were published in grey literature, consisting of a Canadian survey designed to measure serum vitamin D levels and a study in infants presented as an abstract at a conference. Also included were the results of vitamin D tests performed in community laboratories in Ontario between October 2008 and September 2009 (provided by the Ontario Association of Medical Laboratories).
Different threshold levels were used in the studies, thus we reported the percentage of subjects with serum levels of between 25 and 30 nmol/L and between 37.5 and 50 nmol/L. Some studies stratified the results according to factors affecting vitamin D status and two used multivariate models to investigate the effects of these characteristics (including age, season, BMI, vitamin D intake, skin pigmentation, and season) on serum 25(OH)D levels. It’s unclear, however, if these studies were adequately powered for these subgroup analyses.
Study participants generally consisted of healthy, community-dwelling subjects and most excluded individuals with conditions or medications that alter vitamin D or bone metabolism, such as kidney or liver disease. Although the studies were conducted in different parts of Canada, fewer were performed in Northern latitudes, i.e. above 53°N, which is equivalent to the city of Edmonton.
Adults
Serum vitamin D levels of < 25 to 30 nmol/L were observed in 0% to 25.5% of the subjects included in five studies; the weighted average was 3.8% (95% CI: 3.0, 4.6). The preliminary results of the Canadian survey showed that approximately 5% of the subjects had serum levels below 29.5 nmol/L. The results of over 600,000 vitamin D tests performed in Ontarian community laboratories between October 2008 and September 2009 showed that 2.6% of adults (> 18 years) had serum levels < 25 nmol/L.
The prevalence of serum vitamin D levels below 37.5-50 nmol/L reported among studies varied widely, ranging from 8% to 73.6% with a weighted average of 22.5%. The preliminary results of the CHMS survey showed that between 10% and 25% of subjects had serum levels below 37 to 48 nmol/L. The results of the vitamin D tests performed in community laboratories showed that 10% to 25% of the individuals had serum levels between 39 and 50 nmol/L.
In an attempt to explain this inter-study variation, the study results were stratified according to factors affecting serum vitamin D levels, as summarized below. These results should be interpreted with caution as none were adjusted for other potential confounders. Adequately powered multivariate analyses would be necessary to determine the contribution of risk factors to lower serum 25(OH)D levels.
Seasonal variation
Three adult studies evaluating serum vitamin D levels in different seasons observed a trend towards a higher prevalence of serum levels < 37.5 to 50 nmol/L during the winter and spring months, specifically 21% to 39%, compared to 8% to 14% in the summer. The weighted average was 23.6% over the winter/spring months and 9.6% over summer. The difference between the seasons was not statistically significant in one study and not reported in the other two studies.
Skin Pigmentation
Four studies observed a trend toward a higher prevalence of serum vitamin D levels < 37.5 to 50 nmol/L in subjects with darker skin pigmentation compared to those with lighter skin pigmentation, with weighted averages of 46.8% among adults with darker skin colour and 15.9% among those with fairer skin.
Vitamin D intake and serum levels
Four adult studies evaluated serum vitamin D levels according to vitamin D intake and showed an overall trend toward a lower prevalence of serum levels < 37.5 to 50 nmol/L with higher levels of vitamin D intake. One study observed a dose-response relationship between higher vitamin D intake from supplements, diet (milk), and sun exposure (results not adjusted for other variables). It was observed that subjects taking 50 to 400 IU or > 400 IU of vitamin D per day had a 6% and 3% prevalence of serum vitamin D level < 40 nmol/L, respectively, versus 29% in subjects not on vitamin D supplementation. Similarly, among subjects drinking one or two glasses of milk per day, the prevalence of serum vitamin D levels < 40 nmol/L was found to be 15%, versus 6% in those who drink more than two glasses of milk per day and 21% among those who do not drink milk. On the other hand, one study observed little variation in serum vitamin D levels during winter according to milk intake, with the proportion of subjects exhibiting vitamin D levels of < 40 nmol/L being 21% among those drinking 0-2 glasses per day, 26% among those drinking > 2 glasses, and 20% among non-milk drinkers.
The overall quality of evidence for the studies conducted among adults was deemed to be low, although it was considered moderate for the subgroups of skin pigmentation and seasonal variation.
Newborn, Children and Adolescents
Five Canadian studies evaluated serum vitamin D levels in newborns, children, and adolescents. In four of these, it was found that between 0 and 36% of children exhibited deficiency across age groups with a weighted average of 6.4%. The results of over 28,000 vitamin D tests performed in children 0 to 18 years old in Ontario laboratories (Oct. 2008 to Sept. 2009) showed that 4.4% had serum levels of < 25 nmol/L.
According to two studies, 32% of infants 24 to 30 months old and 35.3% of newborns had serum vitamin D levels of < 50 nmol/L. Two studies of children 2 to 16 years old reported that 24.5% and 34% had serum vitamin D levels below 37.5 to 40 nmol/L. In both studies, older children exhibited a higher prevalence than younger children, with weighted averages 34.4% and 10.3%, respectively. The overall weighted average of the prevalence of serum vitamin D levels < 37.5 to 50 nmol/L among pediatric studies was 25.8%. The preliminary results of the Canadian survey showed that between 10% and 25% of subjects between 6 and 11 years (N= 435) had serum levels below 50 nmol/L, while for those 12 to 19 years, 25% to 50% exhibited serum vitamin D levels below 50 nmol/L.
The effects of season, skin pigmentation, and vitamin D intake were not explored in Canadian pediatric studies. A Canadian surveillance study did, however, report 104 confirmed cases1 (2.9 cases per 100,000 children) of vitamin D-deficient rickets among Canadian children age 1 to 18 between 2002 and 2004, 57 (55%) of which from Ontario. The highest incidence occurred among children living in the North, i.e., the Yukon, Northwest Territories, and Nunavut. In 92 (89%) cases, skin pigmentation was categorized as intermediate to dark, 98 (94%) had been breastfed, and 25 (24%) were offspring of immigrants to Canada. There were no cases of rickets in children receiving ≥ 400 IU VD supplementation/day.
Overall, the quality of evidence of the studies of children was considered very low.
Kidney Disease
Adults
Two studies evaluated serum vitamin D levels in Canadian adults with kidney disease. The first included 128 patients with chronic kidney disease stages 3 to 5, 38% of which had serum vitamin D levels of < 37.5 nmol/L (measured between April and July). This is higher than what was reported in Canadian studies of the general population during the summer months (i.e. between 8% and 14%). In the second, which examined 419 subjects who had received a renal transplantation (mean time since transplantation: 7.2 ± 6.4 years), the prevalence of serum vitamin D levels < 40 nmol/L was 27.3%. The authors concluded that the prevalence observed in the study population was similar to what is expected in the general population.
Children
No studies evaluating serum vitamin D levels in Canadian pediatric patients with kidney disease could be identified, although three such US studies among children with chronic kidney disease stages 1 to 5 were. The mean age varied between 10.7 and 12.5 years in two studies but was not reported in the third. Across all three studies, the prevalence of serum vitamin D levels below the range of 37.5 to 50 nmol/L varied between 21% and 39%, which is not considerably different from what was observed in studies of healthy Canadian children (24% to 35%).
Overall, the quality of evidence in adults and children with kidney disease was considered very low.
Clinical Utility of Vitamin D Testing
A high quality comprehensive systematic review published in August 2007 evaluated the association between serum vitamin D levels and different bone health outcomes in different age groups. A total of 72 studies were included. The authors observed that there was a trend towards improvement in some bone health outcomes with higher serum vitamin D levels. Nevertheless, precise thresholds for improved bone health outcomes could not be defined across age groups. Further, no new studies on the association were identified during an updated systematic review on vitamin D published in July 2009.
With regards to non-bone health outcomes, there is no high or even moderate quality evidence that supports the effectiveness of vitamin D in outcomes such as cancer, cardiovascular outcomes, and all-cause mortality. Even if there is any residual uncertainty, there is no evidence that testing vitamin D levels encourages adherence to Health Canada’s guidelines for vitamin D intake. A normal serum vitamin D threshold required to prevent non-bone health related conditions cannot be resolved until a causal effect or correlation has been demonstrated between vitamin D levels and these conditions. This is as an ongoing research issue around which there is currently too much uncertainty to base any conclusions that would support routine vitamin D testing.
For patients with chronic kidney disease (CKD), there is again no high or moderate quality evidence supporting improved outcomes through the use of calcitriol or vitamin D analogs. In the absence of such data, the authors of the guidelines for CKD patients consider it best practice to maintain serum calcium and phosphate at normal levels, while supplementation with active vitamin D should be considered if serum PTH levels are elevated. As previously stated, the authors of guidelines for CKD patients believe that there is not enough evidence to support routine vitamin D [25(OH)D] testing. According to what is stated in the guidelines, decisions regarding the commencement or discontinuation of treatment with calcitriol or vitamin D analogs should be based on serum PTH, calcium, and phosphate levels.
Limitations associated with the evidence of vitamin D testing include ambiguities in the definition of an ‘adequate threshold level’ and both inter- and intra- assay variability. The MAS considers both the lack of a consensus on the target serum vitamin D levels and assay limitations directly affect and undermine the clinical utility of testing. The evidence supporting the clinical utility of vitamin D testing is thus considered to be of very low quality.
Daily vitamin D intake, either through diet or supplementation, should follow Health Canada’s recommendations for healthy individuals of different age groups. For those with medical conditions such as renal disease, liver disease, and malabsorption syndromes, and for those taking medications that may affect vitamin D absorption/metabolism, physician guidance should be followed with respect to both vitamin D testing and supplementation.
Conclusions
Studies indicate that vitamin D, alone or in combination with calcium, may decrease the risk of fractures and falls among older adults.
There is no high or moderate quality evidence to support the effectiveness of vitamin D in other outcomes such as cancer, cardiovascular outcomes, and all-cause mortality.
Studies suggest that the prevalence of vitamin D deficiency in Canadian adults and children is relatively low (approximately 5%), and between 10% and 25% have serum levels below 40 to 50 nmol/L (based on very low to low grade evidence).
Given the limitations associated with serum vitamin D measurement, ambiguities in the definition of a ‘target serum level’, and the availability of clear guidelines on vitamin D supplementation from Health Canada, vitamin D testing is not warranted for the average risk population.
Health Canada has issued recommendations regarding the adequate daily intake of vitamin D, but current studies suggest that the mean dietary intake is below these recommendations. Accordingly, Health Canada’s guidelines and recommendations should be promoted.
Based on a moderate level of evidence, individuals with darker skin pigmentation appear to have a higher risk of low serum vitamin D levels than those with lighter skin pigmentation and therefore may need to be specially targeted with respect to optimum vitamin D intake. The cause-effect of this association is currently unclear.
Individuals with medical conditions such as renal and liver disease, osteoporosis, and malabsorption syndromes, as well as those taking medications that may affect vitamin D absorption/metabolism, should follow their physician’s guidance concerning both vitamin D testing and supplementation.
PMCID: PMC3377517  PMID: 23074397
11.  EVIDENCE FOR A CALCIFICATION PROCESS IN THE TRABECULAR MESHWORK 
Experimental eye research  2008;88(4):738-746.
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.
doi:10.1016/j.exer.2008.11.027
PMCID: PMC2670947  PMID: 19084518
Human trabecular meshwork; Perfused anterior segments, primary HTM cells, Calcification genes; Microarrays; Heat maps
12.  Vitamin K does not prevent soft tissue mineralization in a mouse model of pseudoxanthoma elasticum 
Cell Cycle  2011;10(11):1810-1820.
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.
doi:10.4161/cc.10.11.15681
PMCID: PMC3142464  PMID: 21597330
pseudoxanthoma elasticum; vitamin K; mineralization; Abcc6; mouse
13.  Matrix GLA Protein Function in Human Trabecular Meshwork Cells: Inhibition of BMP2-Induced Calcification Process 
Purpose
The matrix GLA (MGP) gene has been found to be among the 10 most highly expressed genes in the human trabecular meshwork (TM), and its expression is affected by conditions associated with glaucoma. Because MGP protein has been shown to play a key role in inhibiting calcification in cartilage and arterial vessels, MGP’s function in human TM was investigated.
Methods
Perfused TM tissue and primary human TM (HTM) cells originated from donors of nonglaucomatous eyes. MGP mRNA was assayed by relative quantitative and real-time PCR. AdhMGP recombinant adenovirus was generated by bacterial transposition. Western blot analyses were cross-reacted with MGP N-terminal- and conformational-specific antibodies. MGP/ BMP2 colocalization was analyzed by confocal microscopy. γ-Carboxylation activity was measured by incorporation of 14CO2into FLEEL synthetic peptide. Alkaline phosphatase (ALP) activity was used as a marker of osteogenic differentiation and a calcification precursor. Calcification was assessed by measuring direct calcium (o-cresolphthalein). Normalization was conducted with a telomerase probe (genomic DNA).
Results
HTM cells contained high levels of γ-carboxylase activity and were able to convert MGP to its active conformation. Overexpression of MGP in HTM cells reduced ALP activity in a model of BMP2-induced osteogenesis. MGP colocalized intra-cellularly with BMP2. HTM cells aged in culture exhibited increased calcium content, increased ALP, decreased normalized MGP expression and lower γ-carboxylase activity.
Conclusions
MGP protein is active and functions as an inhibitor of BMP2-induced ALP activity in the HTM cells. The human TM may undergo a calcification process with age. Inhibition of the calcification mechanism mediated by MGP could be used to regulate resistance and elevated IOP.
doi:10.1167/iovs.05-1106
PMCID: PMC1592516  PMID: 16505034
14.  Matrix Gla protein deficiency causes arteriovenous malformations in mice 
The Journal of Clinical Investigation  2011;121(8):2993-3004.
Arteriovenous malformations (AVMs) in organs, such as the lungs, intestine, and brain, are characteristic of hereditary hemorrhagic telangiectasia (HHT), a disease caused by mutations in activin-like kinase receptor 1 (ALK1), which is an essential receptor in angiogenesis, or endoglin. Matrix Gla protein (MGP) is an antagonist of BMPs that is highly expressed in lungs and kidneys and is regulated by ALK1. The objective of this study was to determine the role of MGP in the vasculature of the lungs and kidneys. We found that Mgp gene deletion in mice caused striking AVMs in lungs and kidneys, where overall small organ size contrasted with greatly increased vascularization. Mechanistically, MGP deficiency increased BMP activity in lungs. In cultured lung epithelial cells, BMP-4 induced VEGF expression through induction of ALK1, ALK2, and ALK5. The VEGF secretion induced by BMP-4 in Mgp–/– epithelial cells stimulated proliferation of ECs. However, BMP-4 inhibited proliferation of lung epithelial cells, consistent with the increase in pulmonary vasculature at the expense of lung tissue in the Mgp-null mice. Similarly, BMP signaling and VEGF expression were increased in Mgp–/– mouse kidneys. We therefore conclude that Mgp gene deletion is what we believe to be a previously unidentified cause of AVMs. Because lack of MGP also causes arterial calcification, our findings demonstrate that the same gene defect has drastically different effects on distinct vascular beds.
doi:10.1172/JCI57567
PMCID: PMC3148746  PMID: 21765215
15.  Malnutrition, a new inducer for arterial calcification in hemodialysis patients? 
Background
Arterial calcification is a significant cardiovascular risk factor in hemodialysis patients. A series of factors are involved in the process of arterial calcification; however, the relationship between malnutrition and arterial calcification is still unclear.
Methods
68 hemodialysis patients were enrolled in this study. Nutrition status was evaluated using modified quantitative subjective global assessment (MQSGA). Related serum biochemical parameters were measured. And the radial artery samples were collected during the arteriovenous fistula surgeries. Hematoxylin/eosin stain was used to observe the arterial structures while Alizarin red stain to observe calcified depositions and classify calcified degree. The expressions of bone morphogenetic protein 2 (BMP2) and matrix Gla protein (MGP) were detected by immunohistochemistry and western blot methods.
Results
66.18% hemodialysis patients were malnutrition. In hemodialysis patients, the calcified depositions were mainly located in the medial layer of the radial arteries and the expressions of BMP2 and MGP were both increased in the calcified areas. The levels of serum albumin were negatively associated with calcification score and the expressions of BMP2 and MGP. While MQSGA score, serum phosphorus and calcium × phosphorus product showed positive relationships with calcification score and the expressions of BMP2 and MGP.
Conclusions
Malnutrition is prevalent in hemodialysis patients and is associated with arterial calcification and the expressions of BMP2 and MGP in calcified radial arteries. Malnutrition may be a new inducer candidate for arterial calcification in hemodialysis patients.
doi:10.1186/1479-5876-11-66
PMCID: PMC3608064  PMID: 23506394
Arterial calcification; Hemodialysis; Malnutrition; Bone morphogenetic protein 2; Matrix Gla protein
16.  Effects of the Blood Coagulation Vitamin K as an Inhibitor of Arterial Calcification 
Thrombosis research  2008;122(3):411-417.
Introduction
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.
doi:10.1016/j.thromres.2007.12.005
PMCID: PMC2529147  PMID: 18234293
Vitamin K; Arterial calcification; Matrix Gla Protein (MGP); MK4 is an abbreviation for vitamin K4 of the vitamin K2 compounds
17.  Matrix Gla Protein Polymorphism, But Not Concentrations, Is Associated with Radiographic Hand Osteoarthritis 
The Journal of rheumatology  2011;38(9):1960-1965.
Objective
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.
Methods
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.
Results
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).
Conclusion
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.
doi:10.3899/jrheum.100985
PMCID: PMC3200118  PMID: 21724703
MATRIX GLA PROTEIN; POLYMORPHISM; OSTEOARTHRITIS; VITAMIN K
18.  Heat Shock Protein 70 Enhances Vascular Bone Morphogenetic Protein-4 Signaling by Binding Matrix Gla Protein 
Circulation research  2009;105(6):575-584.
Rationale
Matrix Gla protein (MGP) is a calcification inhibitor, which binds and inhibits bone morphogenetic protein (BMP)-2 and -4.
Objective
The objective was to determine if MGP also binds other proteins, which could interfere with its function.
Methods and Results
We transfected bovine aortic endothelial cells with N-terminally FLAG-tagged MGP, and used immunoprecipitation and LC-MS/MS-analysis to identify MGP-binding proteins. Heat shock protein 70 (HSP70), a stress-induced protein expressed in atherosclerotic lesions and soluble in serum, was identified as a novel MGP-binding protein. The interaction between MGP and HSP70 was confirmed by co-immunoprecipitation and chemical crosslinking, and blocked the interaction between MGP and BMP-4. In endothelial cells, HSP70 enhanced BMP-4-induced proliferation and tube formation, and in calcifying vascular cells (CVC), HSP70 enhanced BMP-induced calcium deposition. In addition, HSP70 mediated the procalcific effect of interleukin (IL)-6 on CVC. In apolipoprotein E null mice, a model for atherosclerosis, levels of BMP-4, HSP70, MGP and IL-6 were elevated in the aortic wall. Levels of BMP-4, HSP70 and IL-6 were also elevated in serum, and anti-HSP70 antibodies diminished its procalcific effect on CVC.
Conclusion
HSP70 binds MGP and enhances BMP activity, thereby functioning as a potential link between cellular stress, inflammation and BMP-signaling.
doi:10.1161/CIRCRESAHA.109.202333
PMCID: PMC2779117  PMID: 19661459
Heat shock protein 70; Matrix Gla protein; Bone morphogenetic protein; Protein interaction
19.  Increased levels of the calcification marker Matrix Gla Protein and the inflammatory markers YKL-40 and CRP in patients with type 2 diabetes and ischemic heart disease 
Objective and design
Low grade inflammation is of pathogenic importance in atherosclerosis and in the development of cardiovascular disease (CVD) and type 2 diabetes (T2D). Matrix GLA protein (MGP), an inhibitor of medial calcification of arteries, is increased in patients with atherosclerosis. In the present study levels of markers of calcification (MGP) and inflammation (YKL-40, hsCRP) were evaluated in patients with T2 D and/or ischemic heart disease (IHD).
Materials and methods
The study population consisted of 1) patients with T2D (n = 45); 2) patients with IHD (n = 37); patients with both T2D and IHD (n = 20) and 4) healthy controls (n = 20). Biochemical parameters were measured in venous blood samples.
Results
Levels of MGP, YKL-40 and hsCRP were increased in patients with IHD and/or T2D (p < 0.0001) and patients with T2D and IHD had higher MGP levels (p < 0.001). In multiple linear regression analyses MGP was associated with patient category (r = 0.36, p < 0.001), and HDL-cholesterol levels (r = 0.29, p < 0.001) adjusting for the significant covariates.
Conclusions
In patients with T2D and/or IHD we found increased levels of plasma MGP indicative of a progressing calcification process. This process is paralleled by increased levels of YKL-40 and hsCRP, which most likely reflect the concomitant low grade inflammatory state in these patients
doi:10.1186/1475-2840-9-86
PMCID: PMC3016330  PMID: 21143859
20.  Vitamin K: the effect on health beyond coagulation – an overview 
Food & Nutrition Research  2012;56:10.3402/fnr.v56i0.5329.
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.
doi:10.3402/fnr.v56i0.5329
PMCID: PMC3321262  PMID: 22489224
phylloquinone; menaquinone; cardiovascular disease; osteoporosis; triage
21.  A Matrix Gla Protein Gene Polymorphism is Associated with Increased Coronary Artery Calcification Progression 
Objective
Matrix gla protein (MGP) inhibits arterial and cartilaginous calcification. A Threonine to Alanine (Thr83Ala) polymorphism (codon 83) in MGP is associated with myocardial infarction (MI) and femoral artery calcification. We examined the association of the MGP Thr83Ala polymorphism with quantity and progression of coronary artery calcification (CAC), a non-invasive measure of subclinical coronary atherosclerosis.
Methods and Results
In 605 Epidemiology of Coronary Artery Calcification Study participants, generalized linear mixed models were fit to determine the association of MGP Thr83Ala with CAC quantity and progression. There was a significant additive relationship between MGP Thr83Ala and CAC progression (P=0.001). In the fully-adjusted model, every one Ala83 allele increase was associated with an estimated 1.9% (95% CI: 0.7%, 3.0%) per one-year since baseline larger increase in CAC quantity. A proxy SNP for MGP Thr83Ala (rs6488724) was similarly associated with CAC progression in an independent cohort from the Genetic Epidemiology Network of Arteriopathy (GENOA) Study.
Conclusions
Increased risk of MI associated with MGP ThrAla83 genotype observed elsewhere may be related to faster progression of subclinical coronary atherosclerosis. MGP genotype could be a potential candidate for identifying individuals at increased risk of atherosclerotic disease who would benefit from aggressive primary prevention strategies.
doi:10.1161/ATVBAHA.112.300491
PMCID: PMC3586431  PMID: 23307874
Population; Genetics; Atherosclerosis; Calcium; Imaging
22.  Coordinated expression of matrix Gla protein is required during endochondral ossification for chondrocyte survival 
The Journal of Cell Biology  2001;154(3):659-666.
Matrix Gla protein (MGP) is a 14-kD extracellular matrix protein of the mineral-binding Gla protein family. Studies of MGP-deficient mice suggest that MGP is an inhibitor of extracellular matrix calcification in arteries and the epiphyseal growth plate. In the mammalian growth plate, MGP is expressed by proliferative and late hypertrophic chondrocytes, but not by the intervening chondrocytes. To investigate the functional significance of this biphasic expression pattern, we used the ATDC5 mouse chondrogenic cell line. We found that after induction of the cell line with insulin, the differentiating chondrocytes express MGP in a stage-specific biphasic manner as in vivo. Treatment of the ATDC5 cultures with MGP antiserum during the proliferative phase leads to their apoptosis before maturation, whereas treatment during the hypertrophic phase has no effect on chondrocyte viability or mineralization. After stable transfection of ATDC5 cells with inducible sense or antisense MGP cDNA constructs, we found that overexpression of MGP in maturing chondrocytes and underexpression of MGP in proliferative and hypertrophic chondrocytes induced apoptosis. However, overexpression of MGP during the hypertrophic phase has no effect on chondrocyte viability, but it does reduce mineralization. This work suggests that coordinated levels of MGP are required for chondrocyte differentiation and matrix mineralization.
doi:10.1083/jcb.200106040
PMCID: PMC2196415  PMID: 11489922
endochondral ossification; matrix Gla protein; ATDC5; chondrocytes; apoptosis
23.  Inhibition of Bone Morphogenetic Proteins Protects against Atherosclerosis and Vascular Calcification 
Circulation research  2010;107(4):485-494.
Rationale
The bone morphogenetic proteins (BMP), a family of morphogens, have been implicated as mediators of calcification and inflammation in the vascular wall.
Objective
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.
Conclusion
Our results indicate that BMP signaling is a key regulator of vascular disease, requiring careful control to maintain normal vascular homeostasis.
doi:10.1161/CIRCRESAHA.110.219071
PMCID: PMC2994650  PMID: 20576934
Bone morphogenetic protein; Matrix Gla protein; Atherosclerosis; Vascular calcification; Inflammation
24.  Peptides of Matrix Gla Protein Inhibit Nucleation and Growth of Hydroxyapatite and Calcium Oxalate Monohydrate Crystals 
PLoS ONE  2013;8(11):e80344.
Matrix Gla protein (MGP) is a phosphorylated and γ-carboxylated protein that has been shown to prevent the deposition of hydroxyapatite crystals in the walls of blood vessels. MGP is also expressed in kidney and may inhibit the formation of kidney stones, which mainly consist of another crystalline phase, calcium oxalate monohydrate. To determine the mechanism by which MGP prevents soft-tissue calcification, we have synthesized peptides corresponding to the phosphorylated and γ-carboxylated sequences of human MGP in both post-translationally modified and non-modified forms. The effects of these peptides on hydroxyapatite formation and calcium oxalate crystallization were quantified using dynamic light scattering and scanning electron microscopy, respectively. Peptides YGlapS (MGP1-14: YγEpSHEpSMEpSYELNP), YEpS (YEpSHEpSMEpSYELNP), YGlaS (YγESHESMESYELNP) and SK-Gla (MGP43-56: SKPVHγELNRγEACDD) inhibited formation of hydroxyapatite in order of potency YGlapS > YEpS > YGlaS > SK-Gla. The effects of YGlapS, YEpS and YGlaS on hydroxyapatite formation were on both crystal nucleation and growth; the effect of SK-Gla was on nucleation. YGlapS and YEpS significantly inhibited the growth of calcium oxalate monohydrate crystals, while simultaneously promoting the formation of calcium oxalate dihydrate. The effects of these phosphopeptides on calcium oxalate monohydrate formation were on growth of crystals rather than nucleation. We have shown that the use of dynamic light scattering allows inhibitors of hydroxyapatite nucleation and growth to be distinguished. We have also demonstrated for the first time that MGP peptides inhibit the formation of calcium oxalate monohydrate. Based on the latter finding, we propose that MGP function not only to prevent blood-vessel calcification but also to inhibit stone formation in kidney.
doi:10.1371/journal.pone.0080344
PMCID: PMC3827180  PMID: 24265810
25.  Polymorphisms in MGP gene and their association with lead toxicity 
Toxicology Mechanisms and Methods  2009;19(3):209-213.
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.
doi:10.1080/15376510802488181
PMCID: PMC2736540  PMID: 19730704
Lead toxicity; Blood lead levels; MGP gene polymorphisms; PCR

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