Original Article. Inactive Matrix Gla-Protein Is Associated With Arterial Stiffness in an Adult Population Based Study

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1 Original Article Inactive Matrix Gla-Protein Is Associated With Arterial Stiffness in an Adult Population Based Study Edward Pivin,* Belen Ponte,* Menno Pruijm, Daniel Ackermann, Idris Guessous, Georg Ehret, Yan-Ping Liu, Nadja E.A. Drummen, Marjo H.J. Knapen, Antoinette Pechere-Bertschi, Fred Paccaud, Markus Mohaupt, Cees Vermeer, Jan A. Staessen, Bruno Vogt, Pierre-Yves Martin, Michel Burnier, Murielle Bochud Downloaded from by guest on July 14, 2018 Abstract Increased pulse wave velocity (PWV) is a marker of aortic stiffness and an independent predictor of mortality. Matrix Gla-protein (MGP) is a vascular calcification inhibitor that needs vitamin K to be activated. Inactive MGP, known as desphospho-uncarboxylated MGP (dp-ucmgp), can be measured in plasma and has been associated with various cardiovascular markers, cardiovascular outcomes, and mortality. In this study, we hypothesized that high levels of dp-ucmgp are associated with increased PWV. We recruited participants via a multicenter family-based crosssectional study in Switzerland. Dp-ucMGP was quantified in plasma by sandwich ELISA. Aortic PWV was determined by applanation tonometry using carotid and femoral pulse waveforms. Multiple regression analysis was performed to estimate associations between PWV and dp-ucmgp adjusting for age, renal function, and other cardiovascular risk factors. We included 1001 participants in our analyses (475 men and 526 women). Mean values were 7.87±2.10 m/s for PWV and 0.43±0.20 nmol/l for dp-ucmgp. PWV was positively associated with dp-ucmgp both before and after adjustment for sex, age, body mass index, height, systolic and diastolic blood pressure (BP), heart rate, renal function, low- and high-density lipoprotein, glucose, smoking status, diabetes mellitus, BP and cholesterol lowering drugs, and history of cardiovascular disease (P 0.01). In conclusion, high levels of dp-ucmgp are independently and positively associated with arterial stiffness after adjustment for common cardiovascular risk factors, renal function, and age. Experimental studies are needed to determine whether vitamin K supplementation slows arterial stiffening by increasing MGP carboxylation. (Hypertension. 2015;66: DOI: /HYPERTENSIONAHA ) Online Data Supplement Key Words: matrix Gla-protein calcium-binding protein pulse wave velocity vascular stiffness vitamin K Increased pulse wave velocity (PWV) is the gold standard in assessing stiffening of the aorta. 1 As shown by 3 population studies, all including over thousand participants, PWV refines risk stratification above and beyond traditional cardiovascular risk factors. In multivariable-adjusted analyses, per one standard deviation (SD) increment in PWV, cardiovascular mortality increased by 20% and 60% in a Danish and Taiwanese population samples, respectively. 2,3 The Framingham heart study found that one standard deviation increment of PWV was associated with a 48% increase in cardiovascular disease risk. 4 Matrix Gla-protein (MGP) is acting as a strong inhibitor of soft tissue calcification. 5,6 MGP knockout mice develop massive vascular calcification in their first weeks of life and die within 2 months of vessels rupture. 7 MGP is expressed primarily by chondrocytes and vascular smooth muscle cells. 8,9 To acquire its full calcification inhibitory activity, MGP needs to undergo 2 post-translational modifications: glutamate carboxylation and serine phosphorylation. Both modifications are not exerted completely, so theoretically, 4 different MGP conformations can be found: unmodified and Received January 12, 2015; first decision January 27, 2015; revision accepted April 27, From the Division of Chronic Disease, University Institute of Social and Preventive Medicine (IUMSP) (E.P., F.P., M. Bochud, I.G.), and Department of Medicine, Service of Nephrology and Hypertension, (M.P., M. Burnier), University Hospital of Lausanne (CHUV), Lausanne, Switzerland; Service of Nephrology, Department of Specialties (B.P., P.-Y.M.), and Unit of Population Epidemiology, Department of Community Medicine and Primary Care and Emergency Medicine (I.G.), University Hospital of Geneva, Geneva, Switzerland; University Clinic for Nephrology, Hypertension and Clinical Pharmacology, Inselspital, Bern University Hospital and University of Bern, Bern, Switzerland (D.A., M.M., B.V.); Cardiology, Department of Specialties of Internal Medicine, Geneva University Hospitals, Geneva, Switzerland and Center for Complex Disease Genomics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD (G.E.); Studies Coordinating Centre, Research Unit Hypertension and Cardiovascular Epidemiology, KU Leuven Department of Cardiovascular Sciences, University of Leuven, Leuven, Belgium (Y.-P.L., J.A.S.); R&D Group VitaK, Maastricht University, Maastricht, The Netherlands (N.E.A.D., M.H.J.K., C.V.); and Hypertension Unit, Department of Specialties, University Hospitals of Geneva, Geneva, Switzerland (A.P.-B.). *These authors contributed equally to this work. This article was sent to Marc L. De Buyzere, Guest Editor, for review by expert referees, editorial decision, and final disposition. The online-only Data Supplement is available with this article at /-/DC1. Correspondence to Murielle Bochud, Division des Maladies Chroniques, Institut Universitaire de Médecine Sociale et Préventive, Route de la Corniche 10, CH-1010 Lausanne. Murielle.Bochud@chuv.ch 2015 American Heart Association, Inc. Hypertension is available at DOI: /HYPERTENSIONAHA

2 2 Hypertension July 2015 Downloaded from by guest on July 14, 2018 inactive as desphospho-uncarboxylated MGP (dp-ucmgp), only phosphorylated, only carboxylated, and finally fully modified and active as phosphorylated and carboxylated MGP. 10,11 Carboxylation of MGP is a vitamin K dependent process: vitamin K hydroquinone, a vitamin K metabolite is an active cofactor of the carboxylation of glutamate residues into gammacarboxyglutamate. 12 Active MGP adsorbs to extracellular hydroxyapatite, thus inhibiting crystal growth. 13 It also inhibits vascular smooth muscle cell apoptosis by binding to bone morphogenetic protein 2, a proapoptotic agonist. 14 Two clinical studies investigated the link between dp-ucmgp and CT-detected calcifications. High dp-ucmgp levels were associated with below-knee arterial calcification score among type 2 diabetic patients. 15 A trend in an association between higher circulating dp-ucmgp levels and high coronary artery calcification was observed in postmenopausal healthy women (P=0.06). 16 The objective of this study was to evaluate whether high levels of dp-ucmgp are associated with increased vascular stiffness as measured by aortic PWV in a random adult population sample recruited within the framework of the Swiss Kidney Project on Genes in Hypertension (SKIPOGH). Methods SKIPOGH is a family-based cross-sectional study exploring the role of genes and kidney hemodynamics in BP regulation and kidney function in the general adult population. 17 Study Visits and Definitions Participants were seen in the morning after an overnight fast. BP was measured 5 times in clinic after a resting period of at least 10 minutes in the sitting position with an appropriately sized arm cuff and a nonmercury manual auscultatory sphygmomanometer (A&D UM-101; A&D Company Ltd, Toshima Ku, Tokyo, Japan), 18 according to the guidelines of the European Society of Hypertension. BP and heart rate were the mean of 4 measurements before the recording of PWV. Diabetes mellitus was a self-reported diagnosis or being on treatment with antidiabetic drugs. Cardiovascular disease was considered positive if participants reported a history of stroke or heart disease. Pulse Wave Velocity The arterial waveform was assessed during an 8 second period in the supine position after 15 minutes of rest at the carotid and femoral arteries by applanation tonometry, using a high-fidelity SPC-301 micromanometer (Millar Instruments, Inc., Houston), interfaced with a laptop computer running the SphygmoCor software, version 8.0 or 8.2 (AtCor Medical Pty. Ltd., West Ryde, Australia). In each center, a single board certified nephrologist (B. Ponte, M. Pruijm, D. Ackermann) obtained the arterial waveforms. Using a tape, they measured the distance from the suprasternal notch to the carotid sampling site (distance A) and the distance from the suprasternal notch to the femoral sampling site (distance B). Pulse wave travel distance was distance B minus distance A. Pulse transit time was derived from the average of 10 consecutive beats. Carotid femoral PWV is the ratio of travel distance in meters to transit time in seconds. We assessed intraobserver and interobserver reproducibility in 40 and 15 participants, respectively, according to Bland and Altman s method. 19 The intraobserver and interobserver biases averaged 0.27 m/s (95% confidence interval, ) and 0.43 m/s (95% confidence interval, ), respectively (Figure S1 in the online-only Data Supplement). Laboratory Procedures Venous blood samples were drawn after an overnight fast. Kidney function, serum cholesterol, and glucose were analyzed by standard clinical laboratory methods. Dp-ucMGP was assessed in EDTA plasma using the inaktif MGP isys kit (IDS, Boldon, UK), which is a precommercial dual-antibody test based on the sandwich ELISA developed by VitaK, Maastricht University, The Netherlands. Intraassay variation coefficients are 3.1% for the upper limit of the normal range and 5.4% for the lower limit of the normal range. Inter-assay variation coefficients are 6.9% for the upper limit of the normal range and 13.6% for the lower limit of the normal range (unpublished data). Chronic Kidney Disease Epidemiology Collaboration formula was used to estimate the glomerular filtration rate using serum creatinine. 20 Study Participants From December 2009 to March 2013, 1128 adult participants were recruited in 2 regions (Berne and Geneva) and one city (Lausanne) of Switzerland. A random sample of the inhabitants was drawn using different strategies. Inclusion criteria were (1) having a minimum age of 18 years; (2) being of European ancestry; (3) having at least 1 and ideally 3 first-degree family members willing to participate; and (4) willingness to provide written informed consent. Pregnant or breastfeeding women were not included. The participation rate was 25.6%. The SKIPOGH study was approved by the Institutional Ethical Review Boards of each of the participating university hospitals. Twelve participants had no or insufficient plasma, so 1116 participants in the SKIPOGH study had dp-ucmgp measurements. We excluded 3 participants taking vitamin K antagonists. Ninety-four participants had no PWV measurements for various reasons (obesity, arrhythmias, technical problems, no show). Eighteen participants were removed because of extreme outliers (Table S1) defined as observations lying >3 interquartile ranges away from percentile 25 and 75, leaving 1001 (90%) participants for further analysis. The number of participants at each step is shown in Figure 1. Statistics Statistical analyses were conducted using STATA version 13 (StataCorp, College Station, TX). Continuous and categorical Figure 1. Flowchart of Participants. dp-ucmgp indicates desphospho-uncarboxylated matrix gla-protein; PWV, pulse wave velocity; and SKIPOGH, Swiss Kidney Project on Genes in Hypertension.

3 Pivin et al Arterial Stiffness and Matrix Gla-Protein 3 Downloaded from by guest on July 14, 2018 variables were expressed as mean±sd and as number of participants (%), respectively. Statistical significance was a 2-sided P value of <0.05. Analysis of variance was used to compare variables between tertiles of dp-ucmgp. Medians of dp-ucmgp and PWV were used to separate participants into 4 groups. χ 2 test of independence was applied to look for an association between the 2 variables, and the odds ratio was calculated. We conducted univariate analyses to look for associations between PWV and the following variables of interest: dp-ucmgp, sex, age, body mass index, height, systolic and diastolic pressure, heart rate, renal function (estimated glomerular filtration rate), low- and high-density lipoprotein, smoking status, diabetes mellitus, BP and cholesterol lowering drugs, and history of cardiovascular disease. The technique of multiple imputation was used to estimate missing values for the above-mentioned independent variables, except for dp-ucmgp. We applied multivariable linear mixed regression to determine the independent association of PWV with each variable, with family clusters modeled as a random effect. All models were also adjusted for centers as a fixed effect. Using a backward elimination procedure, removing one by one the variable with the highest nonsignificant P value, only significant variables (P>0.05) were kept in the final model. For continuous variables, results of all linear analyses are presented as β coefficients per standard deviation and associated P values. For continuous variables, β coefficients represent the increase in PWV per one standard deviation increment of the corresponding independent variable. For categorical variables, β coefficients represent the difference in PWV between the 2 categories. Results Mean values were 7.87±2.10 m/s for PWV and 0.43±0.20 nmol/l for dp-ucmgp. 526 (53%) participants were women. Age averaged (±SD) 46.5±17.2 years, body mass index 24.7±4.2 kg/m 2, height 1.71±0.09 m, BP 117.0±16.4 mm Hg systolic and 75.3±9.6 mm Hg diastolic, and heart rate 66.4±10.5 beats per minute. Renal function (estimated glomerular filtration rate) averaged 97.0±17.4 ml/min per 1.73 m 2, low- and high-density lipoprotein 3.12±0.93 and 1.52±0.42 mmol/l, respectively. Glucose mean was 5.15±0.68 mmol/l. Among all participants, 247 (25%) were smoker, 24 (2.4%) had diabetes mellitus, 146 (14.6%) and 37 (3.7%) were on BP or cholesterol lowering drugs, respectively. Ninety-nine (9.9%) participants reported a history of cardiovascular disease. Population characteristics are described in Table 1 according to tertiles of dp-ucmgp and in Table S2 according to sex. Across increasing categories of dp-ucmgp, more participants had diabetes, were on BP lowering drugs, or had a history of cardiovascular disease, whereas fewer reported smoking. Furthermore, age, body mass index, BP, low density lipoprotein and glucose increased with higher category of dp-ucmgp (all P<0.01). Table 1. Baseline Characteristics of Participants by Tertiles of the dp-ucmgp Distribution Characteristic Low Medium High P Value Total dp-ucmgp limits, nmol/l < >0.50 N N with characteristics, % Women 187 (53.9) 176 (51.3) 163 (52.4) (52.5) Current smoker 112 (32.3) 84 (24.5) 51 (16.4) < (24.7) Diabetes mellitus 6 (1.7) 6 (1.8) 14 (4.5) (2.6) BP lowering drugs 23 (6.6) 46 (13.4) 77 (24.7) < (14.6) Cholesterol lowering 8 (2.3) 12 (3.5) 17 (5.4) (3.7) drugs CV disease 20 (5.8) 33 (9.6) 46 (14.7) < (9.9) Mean of characteristic (±SD) PWV, m/s 6.99± ± ±2.36 < ±2.10 Age, y 38.7± ± ±16.1 < ±17.2 Body mass index, kg/m ± ± ±4.4 < ±4.2 Height, m 1.71± ± ± ±0.09 Systolic BP, mm Hg 111±13 117±16 124±18 < ±16 Diastolic BP, mm Hg 73±9 76±10 78±9.0 < ±10 Heart rate, bpm 66.5± ± ± ±10.5 egfr, ml/min per 105±14 98±17 88±18 < ± m 2 LDL, mmol/l 2.90± ± ±0.93 < ±0.93 HDL, mmol/l 1.60± ± ±0.41 < ±0.42 Glucose, mmol/l 4.97± ± ±0.80 < ±0.68 dp-ucmgp, nmol/l 0.23± ± ±0.15 < ±0.20 P-values are for linear trend across tertiles of the dp-ucmgp distribution. Diabetes mellitus was a selfreported diagnosis or being on treatment with antidiabetic drugs. CV disease was considered positive if participants reported a history of stroke or heart disease. BP indicates blood pressure; CV, cardiovascular; Dp-ucMGP, desphospho-uncarboxylated matrix gla-protein; egfr, estimated glomerular filtration rate using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula; HDL, high density lipoprotein; LDL, low density lipoprotein; and PWV, pulse wave velocity.

4 4 Hypertension July 2015 Downloaded from by guest on July 14, 2018 Renal function and high-density lipoprotein decreased with higher category of dp-ucmgp (all P<0.01). In univariate analyses, PWV was significantly associated with all independent variables except heart rate (Table 3). PWV was positively associated with dp-ucmgp (Figure 2); a one SD increase in dp-ucmgp (0.20 nmol/l) was associated with a 0.9 m/s increase in PWV. PWV was positively associated with age, body mass index, low-density lipoprotein, glucose, systolic and diastolic BPs (all P<0.01). It was negatively associated with height (P<0.01), renal function (P<0.01), and high-density lipoprotein (P=0.03). Participants on BP and cholesterol lowering drugs and with diabetes mellitus or cardiovascular disease had a higher PWV (all P<0.01). Current smokers and women had a lower PWV. Distribution of participants according to dp-ucmgp and PWV medians is shown in Table 2. The odd ratio for a PWV >7.4 (m/s) was 3.25 (95% CI, 2.51; 4.21) for higher compared with lower dp-ucmgp levels ( nmol/l). In multivariate analysis (Table 3), we identified the following independent variables as significant positive determinants of PWV: dp-ucmgp, age, body mass index, systolic pressure, heart rate, glucose, diabetes mellitus, and history of cardiovascular disease. Diastolic pressure was a significant negative determinant of PWV. Heart rate was forced into the model for clinical coherence, despite a nonsignificant P value in the univariate analysis. 21 During the backward elimination procedure, independent variables were removed in the following order: HDL, LDL, current smoker, estimated glomerular filtration rate, cholesterol lowering drugs, sex, BP lowering drugs, and finally height (Table S3). Heart rate remained significant in each step of the procedure. In the fully adjusted model, a one SD increase in dp-ucmgp (0.20 nmol/l) was associated with a 0.2 m/s increase in PWV. Beta coefficients are displayed in Table 3 for univariate models, the full model, and the model with only significant independent variables and heart rate. Analysis with pulse pressure and mean pressure (Table S4), with centers as a random effect (Table S5), with extreme outliers (Table S6), with an interaction between age and dp-ucmgp (Table S7) and sensitivity analysis (Table S8 and S9), are presented in the online-only Data Supplement. These additional analyses led to similar results and did not alter our conclusions. Table 2. Distribution of Participants According to dp-ucmgp and PWV Medians Variables Dp-ucMGP, nmol/l Low ( 0.408) High (>0.408) Total PWV, m/s Low ( 7.4) High (>7.4) Total Odd ratio 3.25 (95% CI, 2.51; 4.21), Pearson s chi-squared =82.28, P- value < CI indicates confidence interval; Dp-ucMGP, desphosphouncarboxylated matrix gla-protein; and PWV, pulse wave velocity. Figure 2. Scatterplot of pulse wave velocity (PWV; m/s) and desphospho-uncarboxylated matrix gla-protein (dp-ucmgp; nmol/l). Discussion In this multicentric population-based study, PWV was positively associated with dp-ucmgp in 1001 adults of European descent, independently of other cardiovascular risk factors. To our knowledge, there is no published study that has found a significant association between arterial stiffness and any MGP species in the general adult population. Some studies in selected groups of patients used total MGP or uncarboxylated MGP (ucmgp), without taking phosphorylation status into account, and found no significant association with arterial stiffness. In a study conducted in 144 postmenopausal women, PWV was not associated with total MGP, but was associated with osteoprotegerin, another potential vascular calcification inhibitor. 22 In 81 renal transplant recipients, PWV was again not associated with total MGP. 23 In 120 dialysis patients, PWV was not associated this time with ucmgp. 24 The authors hypothesized a possible sequestration of ucmgp near the calcified areas of arteries because ucmgp levels were also lower in dialysis patients than in healthy control. In 61 children on dialysis, PWV was not associated with ucmgp levels, yet levels were also lower in children on dialysis than in healthy controls. 25 As the molecular weight of MGP is 14 kda, those lower levels cannot be the consequence of the dialysis itself. 26 Dp-ucMGP has a low affinity for calcium in the absence of phosphoserine and gammacarboxyglutamate residues 13 and is probably prone to be set free in the circulation. Moreover, it is elevated in patients at risk of cardiovascular calcification because of end-stage renal disease. 27 Finding a significant association between PWV and dp-ucmgp is a major step forward after all those unsuccessful attempts. It is also a further argument to state that dp-ucmgp probably best reflects MGP bioactivity rather than other MGP species. Whether the observed association reflects a causal effect of MGP on arterial stiffness, or whether MGP is merely a biomarker of vascular vitamin K status, is unclear. We hypothesize that MGP lies on the causal pathway between arterial stiffness and vitamin K status. Vitamin K influences MGP carboxylation. Two randomized double-blind,

5 Pivin et al Arterial Stiffness and Matrix Gla-Protein 5 Table 3. Factors Associated With PWV in Univariate and Multivariate Linear Mixed Regression Models (N=1001) Univariate Models Full Model* Model With Significant Independent Variables Downloaded from by guest on July 14, 2018 Independent Variables placebo-controlled intervention trials bring forward some arguments in favor of a causal role of vitamin K status on arterial stiffness. In the first one, after 3 years of vitamin K1 supplementation, elastic properties of the common carotid artery remained similar in the supplementation group, whereas they worsened in the placebo group. 28 In the vitamin K1 group, the percentage change from baseline for the distensibility coefficient and the compliance coefficient were 0.8% and +2.7%, respectively. In the placebo group, they were 9.6% and 5.9%, respectively. The differences between the 2 groups were significant (P<0.05). The daily supplementation dose of vitamin K1 used (1 mg) was around 10 the recommended dose. The second trial used 180 μg of vitamin K2 also for 3 years. PWV and dpucmgp were measured, among other determinants of vessel wall properties. The results showed a trend in a reduction of PWV (P=0.09) and a significant reduction of dp-ucmgp in the vitamin K2 group. 29 Pearson correlation between baseline PWV and dp-ucmgp was significant at 0.16 (P<0.05), and no multivariable analysis was performed. Participants in both studies were healthy postmenopausal women: 181 SD β P Value β P Value β P Value 95% Confidence Intervals Dp-ucMGP, nmol/l < < < Age, y < < < Body mass index, kg/m < < Height, m < Systolic pressure, mm Hg < < < Diastolic pressure, mm Hg < Heart rate, bpm egfr, ml/min per 1.73 m < LDL, mmol/l < HDL, mmol/l Glucose, mmol/l < Sex (women) Current smoker (yes) < Diabetes mellitus (yes) < BP lowering drugs (yes) < Cholesterol lowering drugs (yes) < CV disease (yes) < All models are adjusted for the center where the PWV was recorded, as a fixed effect. Diabetes mellitus was a self-reported diagnosis or being on treatment with antidiabetic drugs. CV disease was considered positive if participants reported a history of stroke or heart disease. BP indicates blood pressure; CV, cardiovascular; dp-ucmgp, desphospho-uncarboxylated matrix gla-protein; egfr, estimated glomerular filtration rate using the Chronic Kidney Disease Epidemiology Collaboration (CKD-EPI) formula; HDL, high density lipoprotein; LDL, low density lipoprotein; and PWV, pulse wave velocity. *Heart rate was not significant in univariate analysis but forced in the full model for clinical coherence. Model reached at the end of the backward procedure, all steps are presented in Table S3. β, beta coefficient; for continuous variables it represents the increase in PWV per an increase of one standard deviation (SD) of the corresponding independent variable and for categorical variables, the difference of PWV between the 2 categories. P value < in the first trial and 244 in the second one. In a case control study with a mean follow up of 1.2 years, 18 hemodialysis patients on warfarin had a greater increase in PWV than controls not on warfarin. 30 Controls were age-matched (±2 years) and also on hemodialysis. All participants were free of previous cardiovascular disease. Adjusted PWV increased by 0.86 m/s in control and by 2.24 m/s in warfarin group; the difference was significant (P=0.013), despite already high mean PWV at baseline in both groups (12.8 m/s). Finally, a recent Mendelian randomization study found that higher dp-ucmgp predicts noncancer mortality. 31 The Mendelian design strongly supports a causal relationship and also favors the hypothesis that dp-ucmgp is not solely a biomarker of vascular vitamin K status, although both hypotheses could be true. In healthy arteries, most of the MGP occurs in the carboxylated (active) form, and it is found in the tunica media along the elastin fibers. 32 A potential mechanism by which MGP could influence arterial stiffness is via decreased vascular calcifications, considering the known relation between arterial stiffness and the degree of vascular calcification. In

6 6 Hypertension July 2015 Downloaded from by guest on July 14, 2018 rats, deposition of calcium on the medial elastic fibers is responsible for aortic stiffness and an increase in PWV. 33 In the aortic media, calcifications are close to, and stiffen, elastic fibers. 34 Although calcium can be deposited into either the medial or intimal layers of the vasculature, experts hold the view that medial calcifications, rather than intimal calcifications, influence arterial stiffness. 35 Medial calcification is particularly common in diabetes mellitus and advanced renal disease, 36 and uncarboxylated (inactive) MGP is found near calcifications. 32 Circulating levels of dp-ucmgp do not affect vascular calcifications because dp-ucmgp is biologically inactive and not able to bind calcification and bone morphogenetic protein 2. 37,38 However, low levels, as seen with vitamin K supplementation, reflect strong MGP carboxylation and so maximum inhibition of calcification in the arterial wall. 27 Two ongoing randomized trials currently explore whether vitamin K supplementation decreases arterial and valvular calcifications in hemodialysis patients. 39 The results of cardiovascular prevention trials in the hemodialysis population are often controversial, such as with respect to lipid lowering approaches. 40 Irrespective of the outcomes of the ongoing studies in hemodialysis patients, our findings remain of great importance in a general population level. Given the absence of known toxicity of vitamin K supplementation, our data might lead to a large scale intervention in a lower risk population. PWV is an easy, noninvasive, vascular assessment in any future study on the benefits of a primary and earlier supplementation. Furthermore, dp-ucmgp levels enable to monitor compliance on supplementation, adapt vitamin K doses to nutritional intake of the individual, select the best vitamin K type (K1, K2), and choose the right participants. Selecting participants with high dp-ucmgp levels is expected to maximize any benefit and statistical power. In vitro studies and animal models convincingly demonstrated that MGP functions as a vascular calcification inhibitor. 7,10 Humans with absent or nonfunctional MGP develop Keutel syndrome, an autosomal recessive disorder characterized by abnormal cartilage calcification, peripheral pulmonary stenosis, and midfacial hypoplasia. 41 Autopsy of one young suspected Keutel patient, not confirmed by genetic analysis, has shown extensive arterial calcification. 42 By contrast, after a 26-year follow-up, there was no evidence of vascular calcification on chest and abdominal CT in 4 genetically confirmed women with Keutel syndrome. 43 Similarly, cranial CT scan and multislice CT scanning of the coronary arteries revealed no calcification in a 21-year-old men with genetically confirmed Keutel syndrome. 44 Unfortunately, their PWVs were not reported. One hypothesis that could be in line with our results and the absence of calcifications in tomography in Keutel patients is that a high level of inactive MGP leads to an increase in the number, or in the size, of calcifications, but sufficiently scattered in the vessels not to be detectable by tomography. Because to be taken into account in radiological calcification scores, a calcific lesion has to be 1 mm 2 and with a tomographic density of at least 130 Hounsfield units. 45,46 The limitations of the present study include the observational nature of the association, which limits causal inference. We also lack direct assessments of vascular calcification. In our current study, dp-ucmgp was measured at a single time point. However, previous studies showed that dp-ucmgp levels remain unchanged over a 12-week interval. 47 High dp-ucmgp levels could result from an increased synthesis rather than insufficient carboxylation because of lack of vitamin K. Yet, the fact that vitamin K (MK-4) supplementation significantly and massively decreases dp-ucmgp allows us to conclude that it would represent only a small fraction of circulating dp-ucmgp. 27 The participation rate was low mainly because the goal was to enroll at least 3 first-degree family members, yet age and sex distributions are similar to that of the adult Swiss population (Table S10). Although we cannot exclude some level of residual confounding, it is unlikely that unmeasured confounders would eliminate or reverse the observed PWV dp-ucmgp association. Finally, PWV examinations were performed by different operators in different centers. However, linear models were adjusted for the examination centers. Moreover, intra- and interobserver variability showed an excellent reproducibility of the estimates of PWV. Our results shed new light on the role of MGP in arterial stiffness in humans. Because vitamin K therapy lowers dpucmgp levels, 27 these results are a further incentive to conduct additional trials to study the impact of vitamin K supplementation on arterial calcifications and stiffness, in particular in patients at high cardiovascular risk. Perspectives High levels of dp-ucmgp are associated with increased arterial stiffness, measured by PWV, independently of common cardiovascular risk factors in the general adult population. Considering that dp-ucmgp is a marker of vascular vitamin K status, our results are in line with experimental studies in humans showing that vitamin K supplementation stabilizes vascular arterial properties. 28 Randomized clinical trials are needed to further explore whether vitamin K supplementation reduces cardiovascular risk. Acknowledgments We are extremely grateful to the SKIPOGH (Swiss Kidney Project on Genes in Hypertension) study participants. We thank the study nurses involved in the study and recruitment: Marie-Odile Levy, Guler Gök- Sogüt, Ulla Spüchbach, and Dominique Siminski. We thank Sandrine Estoppey for her help in the logistic and database management. Sources of Funding The Swiss National Science Foundation FN33CM The European Union (grants HEALTH-F EU- MASCARA, HEALTHF HOMAGE, and the European Research Council Advanced Research Grant EPLORE) and the Fonds voor Wetenschappelijk Onderzoek Vlaanderen, Ministry of the Flemish Community, Brussels, Belgium (G and G N) support the Research Unit Hypertension and Cardiovascular Research (Leuven, Belgium). Disclosures Nonfinancial support: desphospho-uncarboxylated Matrix Gla- Protein was measured free of charge by VitaK, Maastricht University, The Netherlands. VitaK had no input in the way data were analyzed or presented. VitaK permission had to be obtained before submission of the article. The authors report no conflicts.

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8 8 Hypertension July Munroe PB, Olgunturk RO, Fryns JP, Van Maldergem L, Ziereisen F, Yuksel B, Gardiner RM, Chung E. Mutations in the gene encoding the human matrix Gla protein cause Keutel syndrome. Nat Genet. 1999;21: doi: / Meier M, Weng LP, Alexandrakis E, Rüschoff J, Goeckenjan G. Tracheobronchial stenosis in Keutel syndrome. Eur Respir J. 2001;17: Khosroshahi HE, Sahin SC, Akyuz Y, Ede H. Long term follow-up of four patients with Keutel syndrome. Am J Med. Genet A. 2014;164A: doi: /ajmg.a Cranenburg EC, VAN Spaendonck-Zwarts KY, Bonafe L, Mittaz Crettol L, Rödiger LA, Dikkers FG, VAN Essen AJ, Superti-Furga A, Alexandrakis E, Vermeer C, Schurgers LJ, Laverman GD. Circulating matrix γ-carboxyglutamate protein (MGP) species are refractory to vitamin K treatment in a new case of Keutel syndrome. J Thromb Haemost. 2011;9: doi: /j x. 45. Agatston AS, Janowitz WR, Hildner FJ, Zusmer NR, Viamonte M Jr, Detrano R. Quantification of coronary artery calcium using ultrafast computed tomography. J Am Coll Cardiol. 1990;15: Davila JA, Johnson CD, Behrenbeck TR, Hoskin TL, Harmsen WS. Assessment of cardiovascular risk status at CT colonography. Radiology. 2006;240: doi: /radiol Dalmeijer GW, van der Schouw YT, Magdeleyns E, Ahmed N, Vermeer C, Beulens JW. The effect of menaquinone-7 supplementation on circulating species of matrix Gla protein. Atherosclerosis. 2012;225: doi: /j.atherosclerosis Novelty and Significance Downloaded from by guest on July 14, 2018 What Is New? To our knowledge, there is no published study that finds a significant association between any matrix Gla-protein species and pulse wave velocity as a surrogate marker of arterial stiffness. What Is Relevant? High levels of desphospho-uncarboxylated matrix gla-protein are associated with pulse wave velocity independently of common cardiovascular risk factors in the general adult population. There is ongoing research to prove a beneficial effect of vitamin K in reducing vascular calcifications; our study is a positive preliminary result. Randomized clinical trials are needed to further explore whether vitamin K supplementation reduces cardiovascular risk. Summary In a general adult population, high levels of inactive matrix Glaprotein are positively associated with pulse wave velocity, the gold standard in assessing stiffening of the aorta.

9 Downloaded from by guest on July 14, 2018 Inactive Matrix Gla-Protein Is Associated With Arterial Stiffness in an Adult Population Based Study Edward Pivin, Belen Ponte, Menno Pruijm, Daniel Ackermann, Idris Guessous, Georg Ehret, Yan-Ping Liu, Nadja E.A. Drummen, Marjo H.J. Knapen, Antoinette Pechere-Bertschi, Fred Paccaud, Markus Mohaupt, Cees Vermeer, Jan A. Staessen, Bruno Vogt, Pierre-Yves Martin, Michel Burnier and Murielle Bochud Hypertension. published online May 18, 2015; Hypertension is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX Copyright 2015 American Heart Association, Inc. All rights reserved. Print ISSN: X. Online ISSN: The online version of this article, along with updated information and services, is located on the World Wide Web at: Data Supplement (unedited) at: Permissions: Requests for permissions to reproduce figures, tables, or portions of articles originally published in Hypertension can be obtained via RightsLink, a service of the Copyright Clearance Center, not the Editorial Office. Once the online version of the published article for which permission is being requested is located, click Request Permissions in the middle column of the Web page under Services. Further information about this process is available in the Permissions and Rights Question and Answer document. Reprints: Information about reprints can be found online at: Subscriptions: Information about subscribing to Hypertension is online at:

10 Arterial Stiffness and Matrix Gla Protein -S1- SUPPLEMENTAL MATERIAL INACTIVE MATRIX GLA PROTEIN IS ASSOCIATED WITH ARTERIAL STIFFNESS IN AN ADULT POPULATION-BASED STUDY Short title: Arterial Stiffness and Matrix Gla Protein Edward Pivin*, Belen Ponte*, Menno Pruijm, Daniel Ackermann, Idris Guessous, Georg Ehret, Yan-Ping Liu, Nadja E.A. Drummen, Marjo H.J. Knapen, Antoinette Pechère-Bertschi, Fred Paccaud, Markus Mohaupt, Cees Vermeer, Jan A. Staessen, Bruno Vogt, Pierre-Yves Martin, Michel Burnier, Murielle Bochud Division of chronic disease, University Institute of Social and Preventive Medicine (IUMSP), University Hospital of Lausanne (CHUV), Lausanne, Switzerland (E.P., F.P., M.Boc., I.G.) Service of Nephrology, Department of Specialties, University Hospital of Geneva, Geneva, Switzerland (B.P., P.-Y.M.) Service of Nephrology and Hypertension, University Hospital of Lausanne (CHUV), Lausanne, Switzerland (M.P., M.Bur.) University Clinic for Nephrology, Hypertension and Clinical Pharmacology, Inselspital, Bern University Hospital and University of Bern, 3010 Bern, Switzerland (D.A., M.M., B.V.) Unit of Population Epidemiology, Department of Community Medicine and Primary Care and Emergency Medicine, University Hospital of Geneva, Geneva, Switzerland (I.G.) Cardiology, Department of Specialties of Internal Medicine, Geneva University Hospitals, Geneva, Switzerland and Center for Complex Disease Genomics, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University, Baltimore, MD, USA (G.E.) Studies Coordinating Centre, Research Unit Hypertension and Cardiovascular Epidemiology, KU Leuven Department of Cardiovascular Sciences, University of Leuven, Leuven, Belgium (Y.-P.L., J.A.S.); R&D Group VitaK, Maastricht University, Maastricht, The Netherlands (N.D., M.K., C.V.); Hypertension Unit, University Hospitals of Geneva, Geneva, Switzerland (A.P.-B.) *These authors contributed equally to this work.

11 Arterial Stiffness and Matrix Gla Protein -S2- Material and Methods - Data Supplement This Data Supplement has been provided by the authors to give readers additional information about their work. Extreme outliers The lower and upper outer fences were defined as the first quartile minus three times the interquartile range and the third quartile plus three times the inter-quartile range, respectively. Values smaller than the lower upper fence or bigger than the upper outer fences were defined as extreme outliers. Participants with extreme outliers for PWV or dp-ucmgp, according to the previous definition, were excluded from regression analysis to improve residuals distribution (figure S2). We assessed the distribution of the residuals graphically. 7 participants had PWV higher than 16.3 m/s, the upper outer fence for PWV. 10 participants had dp-ucmgp levels higher than 1.32 nmol/l, the upper outer fence for dp-ucmgp. 1 participant had both dp-ucmgp and PWV above the upper outer fences, so in total 18 participants were removed because of extreme outliers (table S1). No observations were smaller than the lower outer fences. Gender baseline characteristics T-Tests and χ2 tests were performed to compare gender s baseline characteristics for continuous and categorical variables, respectively (table S2). Selection of independent variables in the final models Using a backward elimination procedure, removing one by one the variable with the highest non significant P value, only significant variables (p>0.05) were kept in the final model (table S3). Missing observations for independent variables The technique of multiple imputation was use to estimate missing values for independent variables, except for dp-ucmgp. All independent variables and PWV were used in the imputation models. Linear and logistic regression models were used for the imputation of continuous and categorical variables, respectively. Twenty sets were generated. Only pooled results, using Rubin s combination rules are presented. 34 participants had a total of 63 missing observations that were imputed. The number of missing covariates per independent variable is shown in table S1. Other models used to validate the independent association of PWV with dp-ucmgp We repeated the analysis using mean blood pressure and/or pulse pressure instead of systolic pressure and diastolic pressure (table S4) and using centers as random effects instead of a covariate (table S5). We also performed the analysis including participants with extreme outliers (N=1019): dp-ucmgp was also positively and significantly associated with PWV but residuals were not normally distributed (table S6, figure S2). Beta coefficients of dp-ucmgp stayed positively and significantly associated with PWV in all cases. It was the same when we included an interaction between dp-ucmgp and age (table S7). We performed the analysis in each center separately (table S8) and in women and men separately (table S9). Beta coefficients of dp-ucmgp stayed positively and significantly associated with PWV in all cases, except when we performed the analysis with participants only from center 1, in this case the beta coefficient was positive but not significantly different from zero.