Total Matrix Gla Protein Research Articles

Rationale and Protocol of the ETERNITY-ITA Study: Use of Etelcalcetide for Preserving Vitamin K-Dependent Protein Activity—An Italian Study

Background/Objectives: Chronic kidney disease and mineral bone disorders (CKD-MBD) are frequently associated with an increased risk of both vascular calcifications (VCs) and bone fractures (BFs). The complex pathogenesis of VCs and BFs involves various factors such as calcium overload, phosphate imbalance, and secondary hyperparathyroidism. Key players, such as the vitamin K-dependent proteins (VKDPs) matrix Gla protein (MGP) and bone Gla protein (BGP), have pivotal roles both for VCs and BFs. The VIKI study highlighted that hemodialysis patients treated with calcimimetics had higher levels of total BGP and MGP compared to those untreated, suggesting a potential protective effect of these drugs on BFs and VCs beyond the beneficial effect of reducing PTH levels. Methods: ETERNITY-ITA is a multi-center, comparative effectiveness, observational, longitudinal study that will enroll 160 hemodialysis patients (80 patients treated with Etelcalcetide and 80 age- and sex-matched patients treated with calcitriol or vitamin D analogs). Nephrologists will tailor the target dose of Etelcalcetide on an individual level to achieve the KDIGO PTH target. In the Etelcalcetide-treated group, the addition of calcitriol will be allowed when required by clinical practice (for correction of hypocalcemia). Conclusions: This study will evaluate the real-world effect of Etelcalcetide on VKDP levels, such as BGP and MGP, at 3, 9, and 18 months from baseline. The resulting preservation of vascular and bone health will be assessed for the first time by examining aortic and iliac artery calcifications and vertebral fractures, respectively.

#2035 Use of ETElcalcetidefor pReserving vitamiN K-dependent proteInactivitYITAlian Study (ETERNITY-ITA Study)

Abstract Background and Aims Vascular calcifications (VCs) are prevalent in chronic kidney disease (CKD) and mineral disorders, associated with aortic calcification and increased bone fracture risk. The complex pathogenesis involves factors like calcium overload, phosphate imbalance, and secondary hyperparathyroidism. Key inhibitors, vitamin K-dependent proteins (VKDPs) like matrix Gla protein (MGP) and osteocalcin (BGP), play pivotal roles in VC development. Traditional VC risk reduction focuses on lowering parathyroid hormone, calcium and phosphorus levels. Etelcalcetide, a synthetic peptide activating the calcium-sensing receptor, demonstrates promise in clinical trials. Studies on Vitamin K Italian (VIKI) show that calcimimetic treated hemodialysis patients exhibit higher levels of total BGP and MGP, suggesting a protective effect. These findings underscore the multifactorial nature of VC in CKD, informing refined treatment strategies and targeted pathways for improved outcomes. The proposed study will investigate real world evidence of the effect of Etelcalcetide in preserving active forms of VKDPs among total MGP and total BGP with a resulting reduction of those inactive such as dephosphorylated-undercarboxylated MGP or dp-uc MGP, undercarboxylated BGP or uc-BGP, thus contributing to bone and vascular health in hemodialysis patients (Table 1). Method This is a multi-center comparative effectiveness observational longitudinal study with no predefined interference on drug dosing on the part of the investigators. The study will enroll 160 hemodialysis patients: 80 patients considered in the exposed group (treated with Etelcalcetide) and 80 age and sex matched patients considered in the unexposed group (treated with Calcitriol or vitamin D analogs). The treating nephrologist will base their target dose of Etelcalcetide on an individual-level patient basis in order to achieve the KDIGO PTH target level. In the Etelcalcetide-treated group, the addition of calcitriol is allowed when required by normal clinical practice (for correction of hypocalcemia) (Fig. 1). The primary endpoint is the comparison of the levels of active and non-active forms of VKDP between patients treated with Etelcalcetide and those treated with vitamin D or vitamin D analogues. The two groups will be compared for longitudinal changes in the following biomarkers, measured at baseline and after 3, 9, and 18 months of treatment: total MGP, dephosphorylated-undercarboxylated MGP, total BGP, and undercarboxylated BGP. As secondary outcomes will be evaluated bone vascular markers levels, serum calcification propensity T50 test, anemia markers levels, dialysis routine biomarkers, changes from baseline prevalence VCs (Aorta and Iliac arteries) and vertebral fractures by lateral Dorsal Lumbar spine x-Ray and Bone Mass Density (BMD) by Dual-energy X-ray absorptiometry (DEXA). Results Conclusion

First Comparative Evaluation of Short-Chain Fatty Acids and Vitamin-K-Dependent Proteins Levels in Mother-Newborn Pairs at Birth.

The interplay between vitamin K (vitK) (as carboxylation cofactor, partially produced by the gut microbiota) and short-chain fatty acids (SCFAs), the end-product of fiber fermentation in the gut, has never been assessed in mother-newborn pairs, although newborns are considered vitK deficient and with sterile gut. We collected venous blood from 45 healthy mothers with uncomplicated term pregnancies and umbilical cord blood from their newborns at birth. The concentrations of total SCFAs and hepatic/extra-hepatic vitK-dependent proteins (VKDPs), as proxies of vitK status were assayed: undercarboxylated and total matrix Gla protein (ucMGP and tMGP), undercarboxylated osteocalcin (ucOC), undercarboxylated Gla-rich protein (ucGRP), and protein induced by vitK absence II (PIVKA-II). We found significantly higher ucOC (18.6-fold), ucMGP (9.2-fold), and PIVKA-II (5.6-fold) levels in newborns, while tMGP (5.1-fold) and SCFAs (2.4-fold) were higher in mothers, and ucGRP was insignificantly modified. In mother-newborn pairs, only ucGRP (r = 0.746, p < 0.01) and SCFAs (r = 0.428, p = 0.01) levels were correlated. Conclusions: We report for the first time the presence of SCFAs in humans at birth, probably transferred through the placenta to the fetus. The increased circulating undercarboxylated VKDPSs in newborns revealed a higher vitamin K deficiency at the extrahepatic level compared to liver VKDPs.

The Active Isoforms of MGP Are Expressed in Healthy and Varicose Veins without Calcification.

Matrix Gla protein (MGP), a local inhibitor of tissue mineralization, is associated with vascular calcification. Depending on the carboxylation and phosphorylation status, MGP has active conformations, e.g., carboxylated MGP (cMGP) and phosphorylated MGP (pMGP), but also inactive conformations, e.g., uncarboxylated MGP (ucMGP) and dephosphorylated MGP (dpMGP). Our purpose was to assess the presence of all MGP conformations in healthy veins (HV) and varicose veins (VV), concurrently with the analysis of circulating total MGP (tMGP) before and after the surgical stripping of VV. We collected samples from the great saphenous vein, considered as control group, and tissue from VV, designated as VV group. Plasma levels of tMGP were significantly decreased after the surgical removal of the VV (before 59.5 ± 17.2 vs. after 38.1 ± 11.3, p < 0.001). By using immunohistochemistry staining, we identified local cMGP and pMGP in the control and VV groups, both without calcification, while ucMGP and dpMGP were absent. cMGP was observed in the nucleus and cytoplasm and pMGP in the nucleus of cells belonging to the tunica media, tunica intima and vasa vasorum. Therefore, the active conformations of MGP (cMGP and pMGP) are prevalent in HV and VV without calcification, affirming their anti-calcifying role in veins.

Kidney Function-Dependence of Vitamin K-Status Parameters: Results from the TransplantLines Biobank and Cohort Studies.

High circulating dephosphorylated (dp) uncarboxylated (uc) matrix Gla protein (MGP) and uc osteocalcin (OC) concentrations are regarded as markers of vitamin K-deficiency. However, because MGP and OC are small molecules, they may potentially pass the glomerulus, and their blood concentrations may strongly depend on kidney function. However, many studies with vitamin K-status parameters do not structurally adjust for baseline kidney function, and detailed studies on kidney function-dependence of vitamin K-status markers are lacking. We therefore measured plasma dp-ucMGP using a chemiluminescent assay in 578 kidney transplant recipients (41% females, age 56 ± 13y, 7.5 (3.2 to 13.7)y after transplantation, eGFR 49 ± 17 mL/min/1.73 m2) participating in the prospective TransplantLines Cohort Studies. Additionally, dp-carboxylated MGP, ucOC and carboxylated OC were measured using ELISA in plasma of a subgroup of 60 participants. Finally, dp-ucMGP was measured in a separate cohort of 124 kidney transplant recipients before and three months after kidney transplantation. Dp-ucMGP positively correlated with creatinine, cystatin C, and negatively with eGFR (Spearman’s ρ 0.54, 0.60, and −0.54, respectively, p < 0.001 for all), and each 10 mL/min/1.73 m2 increase in eGFR was associated with a 14.0% lower dp-ucMGP. Additionally, dp-ucMGP strongly declined after kidney transplantation (pretransplantation: 1252 (868 to 1744) pmol/L to posttransplantation: 609 (451 to 914) pmol/L, p < 0.001). Proportions of dp-ucMGP over total MGP and ucOC over total OC were not associated with eGFR. This study highlights that dp-ucMGP is strongly associated with kidney function, and that levels strongly decrease after kidney transplantation. We therefore propose adequate adjustment for kidney function, or the use of kidney function-independent parameters such as proportion of uncarboxylated MGP or OC in the assessment of vitamin K-status in clinical practice and research.

P1645VITAMIN K DEPENDENT PROTEINS AFTER KIDNEY TRANSPLANTATION: RESULTS FROM PROSPECTIVE STUDY

Abstract Background and Aims Two Vitamin K-dependent proteins (VDKPs) link bone and vasculature in CKD-MBD: Bone Gla Protein (BGP) and Matrix Gla Protein (MGP). In ESKD, Vitamin K deficiency is highly prevalent and leads to increased levels of inactive VKDPs (undercaboxylated (ucBGP and dephosphorylated (dp)-uMGP), which are linked to greater risk of fractures and severity of vascular calcification. We hypothesized that kidney transplantation (KT) would improve Vitamin K status and lower levels of inactive VKDPs. Method Between 2014-2017, we conducted a study in 34 patients to assess changes in VKDPs during the 1st year of KT. In a specialized lab we determined VKDPs pre- and 1-year post-KT: total BGP, uc BGP, total MGP, and dp-uc MGP. We determined the prevalence of Vitamin K deficiency based on levels of uc BGP and dp-uc MGP. Results Our cohort had a mean +/- SD age of 48+/-14 years, 32% were female and 97% were Caucasian. 1 year post-KT, there was a decrease in the levels of all VKDPs and the prevalence of Vitamin K deficiency (Table 1 and Figure 1). Patients with greatest severity of Vitamin K deficiency pre-KT had the largest decreases of inactive VDKPs post-KT. Conclusion KT was associated with improvement in Vitamin K status as manifested by decreased levels of inactive VKDPs. These are the first prospective data on VKDPs in CKD patients pre- and post-KT. Studies are needed to assess the impact of improvement in VKDP status after KT on CKD-MBD outcomes.

P1485SEVELAMER USE IS ASSOCIATED WITH DECREASED VITAMIN K LEVELS IN HEMODIALYSIS PATIENTS: RESULTS FROM VITAMIN K ITALIAN (VIKI) STUDY

Abstract Background and Aims Sevelamer (S) is a phosphate binding drug used to treat hyperphosphatemia in patients with CKD. Our aim was to evaluate the hypothesis that the use of (S) could interfere with Vitamin K absorption in hemodialysis (HD) patients of VIKI study. Method We tested this hypothesis in VIKI, a cross-sectional study of 387 hemodialysis patients, we established the prevalence of vitamin K deficiency and to assessed the relationship between vitamin K status, vertebral fractures, vascular calcification. We determined serum concentrations of vitamin 25(OH)D; alkaline phosphatase (ALP); vitamers K1, MK4, MK5, MK6, MK7; osteocalcin (BGP) and Matrix Gla Protein (MGP). We highlighted that MK4 deficiency was the strongest predictor of aortic calcification (OR, 2.82; 95% CI, 1.14–7.01) while vitamin K1 deficiency was the strongest predictor of vertebral fractures fractures (OR: 2.94; 95% CI, 1.38–6.26). Results 163 of 387 patients (42.1%) were treated with Sevelamer. There were no differences in levels of 25(OH)D, K1, MK5, MK6 and MK7 among patients treated with and without Sevelamer. Remarkably, the prevalence of MK4 deficiency was higher in Sevelamer treated patients (13.5% vs 5.4%, p=0.005). Sevelamer treated patients also had higher median levels of ALK (89 UI/L vs 77.5 UI/L, p=0.001) and total BGP (210 mcg/L vs 152 mcg/L, p=0.002) and lower median levels of total MGP (16.4 nmol/L vs 20.3 nmol/L, p=0.037) (Table 1 and Figure 1). In multivariable logistic regression, the odds ratio of MK4 deficiency (dependent variable) in patients treated with compared to without Sevelamer was ∼3-fold higher (OR: 2.64, 95% CI: 1.25-5.58, p=0.011) after adjustment for confounders of Vitamin K levels, including older age, previous myocardial infarction, type of HD, ALP, PTH, MGP, BGP, cholesterol and albumin. Conclusion These data support the hypothesis that Sevelamer could interfere with MK4 absorption in HD patients. Longitudinal interventional studies are needed to prove the causal nature of these associations.

Relationship of matrix Gla protein and vitamin K with vascular calcification in hemodialysis patients

Objective: This study evaluated associations of serum matrix Gla protein (MGP), plasma vitamin K1, and plasma vitamin K2 with coronary artery calcium score (CACS) and cardiovascular disease (CVD) in maintenance hemodialysis (MHD) patients.Methods: Subjects comprised 112 MHD patients aged 30–60 years and 40 age-matched healthy subjects. Total MGP, vitamin K1, vitamin K2, and lipid profile were examined in all subjects; other clinical data, medication use, and CACS were assessed only in MHD patients. Determinants of MGP in all subjects were identified by regression analysis. Factors associated with CACS and CVD in MHD patients were identified by regression analysis and logistic analysis, respectively.Results: Lower plasma levels of vitamin K1 corrected for triglycerides [0.39 (0.24–0.70) vs. 0.77 (0.48–1.34) ng/mg, p < 0.001], higher frequency of plasma vitamin K2 ≤ 0.05 ng/ml (p = 0.23), and higher serum total MGP (288.4 ± 44.2 vs. 159.7 ± 40.6 ng/ml, p < 0.0001) were observed in MHD patients than in healthy controls. Total MGP level was significantly associated with levels of vitamin K1 corrected for triglycerides (p <0 .001) and vitamin K2 ≤ 0.05 ng/ml (p < 0.05) in all subjects. Total MGP level was significantly associated with presence of CVD (p <0 .05), but not CACS, in MHD patients.Conclusion: The end-stage renal disease on hemodialysis is a deficiency state of vitamin K. Total MGP was significantly higher in MHD patients compared to healthy subjects and total MGP was associated with the presence of CVD, but not CACS, in MHD patients.

Vitamin K-Dependent Carboxylation of Matrix Gla Protein Influences the Risk of Calciphylaxis.

Matrix Gla protein (MGP) is a potent inhibitor of vascular calcification. The ability of MGP to inhibit calcification requires the activity of a vitamin K-dependent enzyme, which mediates MGP carboxylation. We investigated how MGP carboxylation influences the risk of calciphylaxis in adult patients receiving dialysis and examined the effects of vitamin K deficiency on MGP carboxylation. Our study included 20 patients receiving hemodialysis with calciphylaxis (cases) and 20 patients receiving hemodialysis without calciphylaxis (controls) matched for age, sex, race, and warfarin use. Cases had higher plasma levels of uncarboxylated MGP (ucMGP) and carboxylated MGP (cMGP) than controls. However, the fraction of total MGP that was carboxylated (relative cMGP concentration = cMGP/[cMGP + uncarboxylated MGP]) was lower in cases than in controls (0.58±0.02 versus 0.69±0.03, respectively; P=0.003). In patients not taking warfarin, cases had a similarly lower relative cMGP concentration. Each 0.1 unit reduction in relative cMGP concentration associated with a more than two-fold increase in calciphylaxis risk. Vitamin K deficiency associated with lower relative cMGP concentration in multivariable adjusted analyses (β=-8.99; P=0.04). In conclusion, vitamin K deficiency-mediated reduction in relative cMGP concentration may have a role in the pathogenesis of calciphylaxis. Whether vitamin K supplementation can prevent and/or treat calciphylaxis requires further study.

OP.1B.01] VITAMIN K DEPENDENT PROTECTION OF RENAL FUNCTION IN MULTI-ETHNIC POPULATION STUDIES

Objective: Vitamin K (VK) dependent carboxylation of matrix Gla protein (MGP) protects the macrocirculation against calcification. Whether glomerular filtration (GFR), a renal microvascular trait, is related to circulating desphospho-uncarboxylated MGP (dp-ucMGP) has not been explored before. We therefore investigated our hypothesis in our Flemish Study on Environment, Genes and Health Outcomes (FLEMENGHO) and sought replication in the South African Study Regarding the Influence of Sex, Age and Ethnicity on Insulin Sensitivity and Cardiovascular Function (SAfrEIC). Design and method: We determined plasma dp-ucMGP in 1166 white Flemish (mean age, 38.2 years) and 714 South Africans (49.2 % blacks; 40.7 years) and total uncarboxylated MGP (t-ucMGP) in Flemish only. dp-ucMGP increases with VK deficiency and t-ucMGP decreases with the extent of arterial calcification. We correlated estimated GFR (CKD-EPI) and stage of chronic kidney disease (KDOQI) with circulating MGP, using multivariable linear and logistic regression. Results: Among Flemish and Africans, GFR averaged 89.5 and 110.9 mL/min/1.73 m2 and dp-ucMGP 3.68 and 7.0 μg/L, respectively. For a doubling of dp-ucMGP, GFR expressed in mL/min/1.73 m2, decreased by 1.46 (p = 0.023) in Flemish and by 2.55 (p = 0.0007) in Africans. Among Flemish, 46.6%, 48.3% and 5.2% were in stage 1, 2 or 3 of chronic kidney disease, while among Africans, these proportions were 80.2%, 18.1%, and 1.7%, respectively. The odds of moving up one stage associated with a doubling of dp-ucMGP were 1.17 (p = 0.033) in Flemish and 1.19 (p = 0.16) in Africans. In Flemish, doubling of dp-ucMGP and t-ucMGP were independently associated with changes in GFR amounting to −1.42 mL/min/1.73 m2 (95% confidence interval [CI], −2.66 to −0.19) and 1.86 (CI, 0.03 to 3.68), respectively. Conclusions: In the general population, GFR decreases and the risk of renal impairment increases with higher dp-ucMGP, a marker of VK deficiency, whereas the opposite is the case for t-ucMGP, a marker of arterial calcification. These findings potentially highlight new avenues for promoting renal health, for instance by vitamin K supplementation.

Matrix Gla-Protein (MGP) Not Only Inhibits Calcification in Large Arteries But Also May Be Renoprotective: Connecting the Dots

Matrix Gla-Protein (MGP) Not Only Inhibits Calcification in Large Arteries But Also May Be Renoprotective: Connecting the Dots

Associations between Thyroid Hormones, Calcification Inhibitor Levels and Vascular Calcification in End-Stage Renal Disease.

IntroductionVascular calcification is a common, serious and elusive complication of end-stage renal disease (ESRD). As a pro-calcifying risk factor, non-thyroidal illness may promote vascular calcification through a systemic lowering of vascular calcification inhibitors such as matrix-gla protein (MGP) and Klotho.Methods and MaterialIn 97 ESRD patients eligible for living donor kidney transplantation, blood levels of thyroid hormones (fT3, fT4 and TSH), total uncarboxylated MGP (t-ucMGP), desphospho-uncarboxylated MGP (dp-ucMGP), descarboxyprothrombin (PIVKA-II), and soluble Klotho (sKlotho) were measured. The degree of coronary calcification and arterial stiffness were assessed by means of cardiac CT-scans and applanation tonometry, respectively.ResultsfT3 levels were inversely associated with coronary artery calcification (CAC) scores and measures of arterial stiffness, and positively with dp-ucMGP and sKlotho concentrations. Subfractions of MGP, PIVKA-II and sKlotho did not associate with CAC scores and arterial stiffness. fT4 and TSH levels were both inversely associated with CAC scores, but not with arterial stiffness.DiscussionThe positive associations between fT3 and dp-ucMGP and sKlotho suggest that synthesis of MGP and Klotho is influenced by thyroid hormones, and supports a link between non-thyroidal illness and alterations in calcification inhibitor levels. However, the absence of an association between serum calcification inhibitor levels and coronary calcification/arterial stiffness and the fact that MGP and Klotho undergo post-translational modifications underscore the complexity of this association. Further studies, measuring total levels of MGP and membrane bound Klotho, should examine this proposed pathway in further detail.

Calcimimetic and vitamin D analog use in hemodialyzed patients is associated with increased levels of vitamin K dependent proteins.

Matrix Gla protein (MGP) and bone Gla protein (BGP) are two vitamin K-dependent proteins (VKDPs) involved in the regulation of vascular calcification (VC). We carried out a secondary analysis of the VIKI study to evaluate associations between drug consumption and VKDP levels in 387 hemodialyzed patients. The VIKI study assessed the prevalence of vitamin K deficiency in hemodialysis patients. We evaluated drug consumption, determined BGP and MGP levels, and verified the presence of any vertebral fractures (VF) and VC by spine radiographs. Total BGP levels were twice as high with calcimimetics versus no calcimimetics (290 vs. 158.5 mcg/L, p < 0.0001) and 69 % higher with vitamin D analogs (268 vs. 159 mcg/L, p < 0.0001). Total MGP was 19 % higher with calcimimetics (21.5 vs. 18.1 mcg/L, p = 0.04) and 54 % higher with calcium acetate (27.9 vs. 18.1 mcg/L, p = 0.003); no difference was found with vitamin D analogs (21.1 vs. 18.3 mcg/L, p = 0.43). Median Total BGP level was 29 % lower in patients with ≥1 VF (151 vs. 213 mcg/L, p = 0.0091) and 36 % lower in patients with VC (164 vs. 262.1 mcg/L, p = 0.0003). In non-survivors, median BGP and MGP were lower, but only for MGP this difference reached the statistical significance (152 vs. 191 mcg/L, p = 0.20 and 15.0 vs. 19.7 mcg/L, p = 0.02, respectively). Pending studies on vitamin K supplementation, calcimimetics, and vitamin D analogs may play a role in preserving vitamin K-dependent protein activity, thus contributing to bone and vascular health in CKD patients.

The association between uncarboxylated matrix Gla protein and lipoprotein-associated phospholipase A2

BackgroundLipoprotein-associated phospholipase A2 (Lp-PLA2) is independently associated with cardiovascular risk, probably via inflammatory activity in sclerotic plaque. We speculated whether Lp-PLA2 has a role in the aetiology of vascular calcifications, estimated from circulating uncarboxylated matrix Gla protein (MGP) species and whether we could find a potential interaction of Lp-PLA2 and MGP in terms of mortality. Materials and MethodsWe examined 798 patients (mean age 65.1 years) with stable vascular disease and followed them in a prospective study. Both, desphospho-uncarboxylated and total MGP (dp-ucMGP or t-ucMGP) were quantified by pre-commercial ELISA assays, developed by VitaK (Maastricht, The Netherland) ResultsLp-PLA2 activity was independently positively associated with desphospho-uncarboxylated MGP (dp-ucMGP) [β coeff=0.098, p=0.006]. 1SD of Lp-PLA2 activity was associated with 37% increased risk (p=0.001) of elevated dp-ucMGP (≥977pmol/L, top quartile). In the Cox proportional hazard model adjusted for conventional risk factors, the patients in the highest quartile of dp-ucMGP or lowest quintile of total-uncarboxylated ucMGP (<2660nmol/L) had higher risk of all-cause mortality [HRR 2.79 (95% CI 1.97–3.94) and HRR 1.69 (95% CI 1.18–2.42), respectively]. We observed no effect of high Lp-PLA2 activity (≥195nmol/min/mL) on total mortality. ConclusionsWe assume that Lp-PLA2 is involved in vascular calcification and that dp-ucMGP is a more appropriate biomarker of residual risk than Lp-PLA2 itself.

Bone metabolism regulators and arterial stiffness in postmenopausal women

BackgroundOsteoprotegerin (OPG), osteopontin (OPN) and matrix Gla protein (MGP) are markers of bone metabolism but they are also involved in vascular calcification. However, their precise role is not completely understood. Arterial stiffness is considered an independent predictor of cardiovascular events and it may be one of the causes of the increased cardiovascular risk associated with postmenopausal status. Medial and intimal calcification may increase arterial stiffness. The aim of our study was to assess the relationship of OPG, OPN and MGP with aortic pulse wave velocity (aPWV) as a marker of arterial stiffness in postmenopausal women. Materials and methodsCirculating OPG, OPN and serum total MGP were measured in 144 postmenopausal women using the enzyme-linked immunosorbent assay method. Aortic PWV was determined by an oscillometric method. ResultsOsteoprotegerin correlated with age (p<0.001, r=0.27), aPWV (p<0.001, r=0.32) and hypersensitive C reactive protein (hsCRP) (p<0.001, r=0.37), OPN correlated directly with hsCRP (p<0.001, r=0.39) and inversely with high density lipoprotein cholesterol (p=0.02, r=−0.02). No significant association was found between total MGP and clinical, biochemical and vascular parameters. The correlation between OPG and aPWV persisted even after the adjustment for various potential confounders (p=0.02, r=0.19). In multiple regression analysis in the whole study population the most important predictors of aPWV were OPG (β=0.230, p=0.006), hsCRP (β=0.212, p=0.01) and systolic blood pressure (β=0.163, p=0.04). After exclusion of patients treated with statins the independent predictors were hsCRP (β=0.275, p=0.005) and OPG (β=0.199, p=0.04). ConclusionCirculating OPG, but not OPN and total MGP, is associated with aPWV and may be a marker of the increased arterial stiffness and cardiovascular risk in postmenopausal women.

Abstract P397: Circulating Species of Matrix Gla Protein and the Risk of Vascular Calcification in Healthy Women

Background: Matrix Gla protein (MGP) is an inhibitor of vascular calcification that requires vitamin K for its carboxylation. Circulating MGP occurs as various species with different conformations depending on carboxylation and phosphorylation. Total uncarboxylated MGP (t-ucMGP) is the sum of non-phosphorylated and phosphorylated uncarboxylated MGP (dp-ucMGP and p[[Unable to Display Character: &amp;#8209;]]ucMGP) and mainly consists of p-ucMGP. Due to its phosphorylated group p-ucMGP has a strong calcium-binding capacity irrespective of the Gla content. In high risk populations, high levels of t[[Unable to Display Character: &amp;#8209;]]ucMGP were associated with decreased calcification, while high levels dp-ucMGP were associated with low vitamin K status and increased vascular calcification. These relations have never been investigated in a healthy population. Objective: This prospective study investigates the association between circulating matrix Gla protein species and vascular calcifications among postmenopausal women. Design: In a prospective cohort of 571 healthy women, MGP levels were measured by ELISA techniques at baseline. Vitamin K intake was estimated from a validated food frequency questionnaire. Calcification was measured in the coronary arteries, aortic valve, mitral valve, and aortic arch by multi-detector computed tomography after 8.5 years follow-up. Coronary artery calcification (CAC) was defined present if Agatston score was ≥10 and the calcification score was calculated as the sum of the calcified areas. Multivariate adjusted relative risks (RR) and odds ratios (OR) were estimated using modified Poisson regression and multinomial logistic regression per standard deviation of MGP species and the no calcification group was used as reference. Results: Of the women, 24% had no calcification while 5% had calcification in all areas. High dp-ucMGP levels, reflecting poor vitamin K status, were (p=0.08) associated with more CAC (RR SD 1.07;95%CI 0.99-1.15) and calcification areas (OR (4 areas vs no calcification) 1.49; 95%CI 0.95-2.35, p=0.09). High t-ucMGP levels were associated with less calcification (OR (4 areas vs no calcification) 0.63; 95%CI 0.36-1.10, p=0.10). Desphospho-carboxylated MGP was not associated with calcification. High phylloquinone intake was not associated with the number of calcified areas OR (4 areas vs no calcification) 1.14 (95%CI: 0.72-1.81, p=0.57). In contrast, higher menaquinones intake was associated with less calcification areas (OR (4 areas vs no calcification) 0.39; 95%CI: 0.21-0.74, p=0.004). Conclusion: High dp-ucMGP levels, reflecting a poor vitamin K status, may be associated with more calcification, also in a healthy population. High menaquinones intake was associated with less calcification. Consistent with high-risk populations, high t-ucMGP levels seem to associated with fewer calcification areas.

High Prevalence of Vertebral Fractures Assessed by Quantitative Morphometry in Hemodialysis Patients, Strongly Associated with Vascular Calcifications

Few studies have provided information on the prevalence of vertebral fractures (VFs) and their risk factors in hemodialysis patients. A multicenter, cross-sectional, observational study was carried out to assess the prevalence of VFs and vascular calcifications (VCs) in 387 hemodialysis patients (mean age 64.2 ± 14.1 years, 63 % males) and in a control group of 51 osteoporotic subjects. Biochemical tests included 25(OH) vitamin D, bone Gla protein (total and undercarboxylated), and total matrix Gla protein. Vertebral quantitative morphometry was carried out centrally for the detection of VF, defined as reduction by ≥20 % of one of the vertebral body dimensions. In the same radiograph, aortic and iliac VC scores were calculated. Prevalence of VF was 55.3 % in hemodialysis patients and 51.0 % in the control group. Multivariate analysis disclosed that male gender (59.8 vs. 47.6 %, p = 0.02; OR = 1.78, 95 % CI 1.15-2.75) and age (mean ± SD 66.7 ± 13.1 vs. 61.0 ± 14.7 years, p < 0.001; OR = 1.03, 95 % CI 1.01-1.05) were significantly associated with VF. The prevalence of aortic VC was significantly higher in hemodialysis patients than in controls (80.6 vs. 68.4 %, p = 0.001). The factors with the strongest association with VC, apart from atrial fibrillation, were serum 25(OH)vitamin D levels below 29 ng/mL for aortic VC (OR = 1.85, 95 % CI 1.04-3.29) and VF both for aortic (OR = 1.77, 95 % CI 1.00-3.14) and iliac (OR = 1.96, 95 % CI 1.27-3.04) VC. In conclusion, the prevalence of VF, especially in males, and VC, in both genders, is high in hemodialysis patients. VF is associated with VC. Vitamin D deficiency is also associated with VC. Further longitudinal studies are warranted to investigate fractures in renal patients.

584 Circulating Matrix GLA Protein and Faster Progression Rate of Aortic Stenosis - a Substudy of the Astronomer Trial

In a previous substudy of the ASTRONOMER trial, we reported that metabolic syndrome (MetS) is associated with faster progression of aortic stenosis (AS). Recent studies suggested that dysregulation of mineral metabolism or its modulators could play a role in the pathogenesis of AS. The carboxylated form of the Matrix Gla Protein (MGP) is an inhibitor of ectopic calcification. The objective of this study was to determine the association between circulating levels of the total (i.e. carboxylated and non carboxylated) MGP and AS progression rate. Among the 269 patients randomized in ASTRONOMER, mean follow-up time 3.4 ±1.3 years, 217 had baseline measurement of circulating level of total MGP. None of the patient had hypercholesterolemia, diabetes, or coronary artery disease at baseline. However, 29% had systemic hypertension and 28% had MetS. Hemodynamic progression rate of AS was measured with the use of the annualized increase in peak aortic jet velocity (Vpeak). In the whole cohort, baseline MGP level was significantly associated with AS progression rate in univariate analysis (r=0.15; p=0.03) but not in multivariate analysis (p=NS). There was a significant interaction (p=0.03) between MGP level and age with respect to its relationship with AS progression rate. In the subset of patients with age<57 years (median value), higher MGP level was significantly associated with faster AS progression rate (r=0.22; p=0.02) whereas no such relationship was observed in older patients. After adjustment for gender, hypertension, LDL-chol, creatinin, baseline valve calcification and Vpeak, and status of randomization (statin vs. placebo), higher MGP level (p=0.008) and MetS (p=0.001) were independent predictors of faster AS progression in younger patients. Baseline level of MGP was positively related to age (p=0.03), LDL-chol (p=0.02), oxidized-LDL (p=0.005), glycaemia (p=0.001), and HOMA-IR index (p=0.05). In multivariate analysis, oxidized-LDL (p=0.03) and glycaemia (p=0.01) were the only two factors independently associated with circulating MGP levels. This study reports an association between higher circulating levels of total MGP and faster progression of AS in the younger population. These findings suggest that valvular calcification may lead to increased MGP expression, perhaps in a feedback attempt to physiologically reduce bone-like formation of calcium deposits in the valve. Further studies with measures of both carboxylated and uncarboxylated forms of MGP in the blood as well as in the valvular tissues are needed to clarify the exact role of the MGP system in the pathogenesis of AS.

Circulating matrix Gla protein is associated with coronary artery calcification and vitamin K status in healthy women

Matrix Gla protein (MGP) is a vitamin K-dependent protein and an inhibitor of vascular calcification. Vitamin K is required for the carboxylation of MGP and can thereby reduce calcification. Circulating MGP species with different conformations have been investigated as markers for coronary artery calcification (CAC). In high-risk populations, high total uncarboxylated MGP (t-ucMGP) was associated with decreased CAC, while high non-phosphorylated uncarboxylated MGP (dp-ucMGP) was associated with a poor vitamin K status. This cross-sectional study investigated the association of MGP species with CAC, vitamin K status among 200 healthy women. Circulating dp-ucMGP, t-ucMGP and, non-phosphorylated carboxylated MGP (dp-cMGP) levels were measured by ELISA techniques and Agatston score by multi-detector computed tomography. The ratio of uncarboxylated to carboxylated osteocalcin was used as proxy of vitamin K status. A borderline significant (P=.06) association between higher circulating dp-ucMGP levels and high CAC was observed (β=0.091, 95% CI−0.01; 0.19). In the entire study population, high t-ucMGP levels tended to be associated (P=.09) with lower CAC (β=−0.36, 95% CI:−0.78; 0.06). This association strengthened amongst women with CAC to a significant relation between high t-ucMGP levels and lower CAC (β=−0.55, 95% CI−1.01;−0.10). Dp-cMGP was not associated with CAC. Low vitamin K-status was associated with high dp-ucMGP concentrations (β=0.138, 95% CI 0.09; 0.19) but not with other MGP species. These results show that dp-ucMGP may serve as a biomarker of vitamin K status. Circulating dp-ucMGP and t-ucMGP may serve as markers for the extent of CAC, but these findings need to be confirmed.

Abstract P071: The Effect of Menaquinone -7 Supplementation on Circulating Species of Matrix-Gla Protein

Background: Menaquinones intake is associated with a reduced risk of coronary calcification and coronary heart disease. This association may be explained by increased carboxylation of matrix-Gla protein (MGP) by menaquinones. However, the effect of menaquinone supplementation on MGP carboxylation has not been investigated to date. Objective: To investigate if 180 µg/day and 360 µg/day menaquinone supplementation may increase carboxylation of MGP. Design: A randomized, double-blind, placebo-controlled trial was performed. Sixty subjects aged 40–65 years were randomly allocated to supplementation of 180 μg/d, 360 μg/d of menaquinone-7 (MK-7) or placebo during 12 weeks. At baseline, after 4 and 12 weeks, desphospho-uncarboxylated MGP (dp-ucMGP), desphospho-carboxylated MGP (dp-cMGP) and total uncarboxylated MGP (t-ucMGP) were measured by ELISA techniques. Furthermore, uncarboxylated osteocalcin (ucOC), carboxylated osteocalcin (cOC) and various cardiovascular risk factors were measured. The ratio of ucOC to cOC was used as proxy of vitamin K status. Results: Dp-ucMGP, decreased significantly and dose-dependently in the 180 μg and 360 mg MK-7 supplementation groups ( P time*treatment &lt;0.001) by 31% (400 pM to 276 pM) and 46% (391 pM to 210 pM) respectively after 12 weeks, while dp-ucMGP circulation levels remained unchanged after placebo treatment. The osteocalcin ratio also decreased significantly and dose-dependently in the 180 μg and 360 mg MK-7 treatment ( P time*treatment &lt;0.001) by 57% (0.44 to 0.19) and 74% (0.42 to 0.11) respectively after 12 weeks, while osteocalcin ratio remained unchanged after placebo treatment, showing improved vitamin K status and indicating good compliance to the study treatment. Changes over time of dp-cMGP (p=0.42) and t-ucMGP (p=0.23) levels did not differ between treatment arms. Also the prothrombin time did not differ between treatments arms ( P time*treatment=0.92). Furthermore, changes over time in other risk factors of cardiovascular disease like blood lipid profile or blood pressure did not differ between treatments. Conclusions: Supplementation with MK-7 decreased dp-ucMGP with 31% to 46%. Like the osteocalcin ratio, dp-ucMGP can be used as a sensitive marker of vitamin K status. MK-7 supplementation has no effect on dp-cMGP and t-ucMGP concentrations.