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Vol. 45, No. 5, 2008
Issue release date: August 2008

Free access is sponsored by an educational grant of the European Society for Microcirculation
J Vasc Res 2008;45:427–436
(DOI:10.1159/000124863)

The Circulating Inactive Form of Matrix Gla Protein (ucMGP) as a Biomarker for Cardiovascular Calcification

Cranenburg E.C.M.a, b · Vermeer C.a, b · Koos R.d · Boumans M.-L.a · Hackeng T.-M.a · Bouwman F.G.c · Kwaijtaal M.a · Brandenburg V.M.e · Ketteler M.f · Schurgers L.J.a, b
aVitaK, bCardiovascular Research Institute CARIM, and cDepartment of Human Biology NUTRIM, Maastricht University, Maastricht, The Netherlands; Departments of dCardiology and eNephrology and Clinical Immunology, RWTH University Hospital Aachen, Aachen, fKuratorium für Heimdialyse, Dialysis Center, Würselen, Germany
email Corresponding Author

Abstract

Objective: Matrix γ-carboxyglutamate (Gla) protein (MGP) is a vitamin K-dependent protein and a strong inhibitor of vascular calcification. Vitamin K deficiency leads to inactive uncarboxylated MGP (ucMGP), which accumulates at sites of arterial calcification. We hypothesized that as a result of ucMGP deposition around arterial calcification, the circulating fraction of ucMGP is decreased. Here we report on the development of an ucMGP assay and the potential diagnostic utility of monitoring serum ucMGP levels. Methods and Results: An ELISA-based assay was developed with which circulating ucMGP can be determined. Serum ucMGP levels were measured in healthy subjects (n = 165) and in four patient populations; patients who underwent angioplasty (n = 30), patients with aortic stenosis (n = 25), hemodialysis patients (n = 52), and calciphylaxis patients (n = 10). All four patient populations had significantly lower ucMGP levels. In angioplasty patients and in those with aortic stenosis, some overlap was observed with the control population. However, in the hemodialysis and calciphylaxis populations, virtually all subjects had ucMGP levels below the normal adult range. Conclusion: Serum ucMGP may be used as a biomarker to identify those at risk for developing vascular calcification. This assay may become an important tool in the diagnosis of cardiovascular calcification.


 Outline


 goto top of outline Key Words

  • Arteries
  • Atherosclerosis
  • Calcification
  • Extracellular matrix
  • Matrix Gla protein
  • Vitamin K

 goto top of outline Abstract

Objective: Matrix γ-carboxyglutamate (Gla) protein (MGP) is a vitamin K-dependent protein and a strong inhibitor of vascular calcification. Vitamin K deficiency leads to inactive uncarboxylated MGP (ucMGP), which accumulates at sites of arterial calcification. We hypothesized that as a result of ucMGP deposition around arterial calcification, the circulating fraction of ucMGP is decreased. Here we report on the development of an ucMGP assay and the potential diagnostic utility of monitoring serum ucMGP levels. Methods and Results: An ELISA-based assay was developed with which circulating ucMGP can be determined. Serum ucMGP levels were measured in healthy subjects (n = 165) and in four patient populations; patients who underwent angioplasty (n = 30), patients with aortic stenosis (n = 25), hemodialysis patients (n = 52), and calciphylaxis patients (n = 10). All four patient populations had significantly lower ucMGP levels. In angioplasty patients and in those with aortic stenosis, some overlap was observed with the control population. However, in the hemodialysis and calciphylaxis populations, virtually all subjects had ucMGP levels below the normal adult range. Conclusion: Serum ucMGP may be used as a biomarker to identify those at risk for developing vascular calcification. This assay may become an important tool in the diagnosis of cardiovascular calcification.

Copyright © 2008 S. Karger AG, Basel


goto top of outline Introduction

Vitamin K-dependent proteins are present in blood and in a wide variety of tissues throughout the body; they exert a broad range of functions, for example as blood coagulation factors in hemostasis [1]. In recent years, one of these proteins has gained increasing attention: matrix γ-carboxyglutamate (Gla) protein (MGP), a potent inhibitor of calcification present in cartilage and the vessel wall, where it is synthesized by chondrocytes and vascular smooth muscle cells (VSMCs), respectively [2, 3]. MGP contains 9 glutamate residues, 5 of which may be γ-carboxylated in a posttranslational step catalyzed by the vitamin K-dependent γ-glutamate carboxylase [1]. Deficiency of vitamin K, either due to nutritional deficiency or the use of coumarin-derivatives, results in undercarboxylation of MGP and impairment of its biological function [4].

With immunohistochemical techniques based on monoclonal antibodies specifically recognizing carboxylated and uncarboxylated MGP (cMGP and ucMGP, respectively), we have recently demonstrated the massive accumulation of ucMGP in atherosclerotic lesions and areas of calcification [5]. This has been a consistent finding in several studies. Price et al. [ 6] reported the accumulation of ucMGP at sites of arterial calcification in rats treated with vitamin D and warfarin and Sweatt et al. [ 7] showed that the aortic wall of aging rats contained elevated concentrations of ucMGP. It was proposed that ucMGP could bind to apatite present in calcifying tissues irrespective of its Gla-residues, which was supported by the fact that thermal decarboxylation of MGP in vitro did not affect its affinity for hydroxyapatite [8].

The ability of MGP to inhibit calcification was demonstrated in transgenic MGP-deficient mice. The MGP null mice were born normally but all developed severe medial arterial calcifications and subsequent vascular ruptures leading to death within 6 weeks [9]. This vascular phenotype could only be rescued if MGP was restored locally in the VSMC and if MGP was γ-carboxylated [10], suggesting that MGP is a local inhibitor of vascular calcification and that its function depends on the presence of Gla residues, respectively. Gla residues are able to bind calcium both in solution and in calcium salt crystals, which is thought to be associated with the ability of MGP to inhibit crystal growth [8, 11]. Moreover, it has been suggested that its mechanism of action is based on the binding of MGP to BMP-2 [12, 13], a bone morphogenetic protein. MGP can also bind to vitronectin, a multifunctional protein present in plasma and extracellular matrix [14]. Nishimoto et al. [ 14] proposed that this binding may assist cells in developing tissues to migrate and differentiate.

In humans, loss-of-function mutations in the MGP gene result in the Keutel syndrome [15]: a recessive disorder characterized by abnormal cartilage calcification, peripheral pulmonary artery stenoses, brachytelephalangism and neural hearing loss [16]. Post-mortem examination of one of the first Keutel patients described has revealed calcification of arteries throughout the body [17]. The propensity to develop arterial calcification is present in a wide range of other patient populations including patients with diabetes, atherosclerosis, and renal dysfunction. Additionally, patients using oral anticoagulants have significantly increased aortic valve calcification and coronary artery calcification [18, 19]. Patients with moderate to severe arterial calcification (as assessed by MSCT) have an unfavorable prognosis compared to patients with no or mild calcification [20,21,22]. Therefore, the measurement of biomarkers which can predict or reflect the disease becomes of substantial importance.

The first commercially available MGP assay does not discriminate between carboxylated and uncarboxylated MGP [23]. Using this assay, we have reported increased as well as decreased circulating MGP levels in patients suffering from various forms of atherosclerotic disease [5, 23, 24]. A correlation between MGP levels and coronary artery calcification (CAC) was found in subjects with suspected coronary artery disease [25]; however, no correlation with CAC could be established in patients with chronic kidney disease (CKD) [26]. Additionally, in CKD patients increased levels have been described [24], as well as no significant difference in MGP levels between these patients and control subjects [27]. Thus, although this assay performs well, results are sometimes conflicting and the strong overlap between serum MGP levels in patients and the normal range does not allow its use for individual diagnosis.

We have developed a competitive enzyme-linked immunosorbent assay (ELISA) which specifically recognizes ucMGP. The accumulation of MGP in its uncarboxylated form at sites of arterial calcification and its probable high affinity for hydroxyapatite [5, 7, 8], suggest that ucMGP is not set free into the circulation from calcified arteries. We hypothesized that in the presence of arterial calcification (arterial medial calcification or in atherosclerotic lesions), the circulating fraction of ucMGP is decreased. To test our hypothesis and to evaluate the po- tential diagnostic utility of the assay, we have measured ucMGP in serum and plasma from different patient cohorts.

 

goto top of outline Materials and Methods

goto top of outline Subjects

Apparently healthy subjects, 91 males and 74 females, were recruited from the general Maastricht population for the assessment of the normal range and reference. Patient samples were acquired from a total of 117 subjects: 52 hemodialysis patients and 10 calciphylaxis patients from the Department of Nephrology and Clinical Immunology of the University Hospital Aachen, 25 aortic stenosis patients from the Department of Cardiology of the University Hospital Aachen, and 30 patients with a history of recent angioplasty from the University Hospital Maastricht. An exclusion criterion for both the reference population and the patient groups was the use of coumarin derivatives. In addition, we measured the ucMGP level in serum of a patient with Keutel syndrome (kind gift of Dr. M. Meier, Clinic of Pneumology, Immenhausen, Germany). The study was approved by the local medical ethics committee, and informed consent was obtained from all participants.

goto top of outline Serum Handling

Blood was collected by venipuncture in serum tubes (10 ml; BD Vacutainer Systems, Plymouth, UK) and in sodium citrate (10 ml; BD Vacutainer Systems) and stored for 20 min at room temperature before centrifugation (15 min 1,580 g). Serum and plasma were subsampled in 250-μl aliquots and frozen at –80°C until testing.

goto top of outline Materials

All chemicals and reagents described below were of analytical grade or better and obtained from commercial suppliers. Synthetic peptides homologous to the uncarboxylated MGP sequences 35–49 and 35–54 (designated as ucMGP35–49 and ucMGP35–54, respectively) and carboxylated MGP sequences 35–49 and 35–54 (cMGP35–49 and cMGP35–54) were made by Pepscan (Lelystad, The Netherlands). All peptides were purified and analyzed by LC/MS (ion spray, positive ion detection) and their purity level was >98% for all peptides. Synthetic full-length MGP, cMGP [28] or ucMGP was used for antibody screening and to define assay specificity. The tracer for the assay was prepared by biotinylating the noncarboxylated 35–54 peptide (ucMGP35–54) using the EZ-Link Maleimide PEO2-Biotin kit according to the manufacturer’s instructions (Pierce, Etten-Leur, the Netherlands).

goto top of outline ucMGP Assay

The monoclonal antibody against ucMGP used for serum measurements was provided by VitaK BV (Maastricht, The Netherlands);the same antibody has been used previously for immunohistochemical staining [5, 29]. The monoclonal antibody (IgG1 isotype, designated as mAb-ucMGP) was raised against the human ucMGP Gla-domain (residues 35–49), and selected for its specificity towards ucMGP using standard techniques [5]. Polyclonal rabbit-anti-mouse IgG (Dako, Heverlee, Belgium) was diluted 2,000-fold in carbonate buffer (0.1 M sodium carbonate, pH 9.6) and used for the coating of the microtiter plate (100 μl/well). After incubation for 1.5 h at 37°C, the remaining protein-binding sites were blocked with 200 μl of blocking buffer (2% HNBSA consisting of HEPES-NaCl buffer (175 mM NaCl, 25 mM HEPES, pH 7.7)) and bovine serum albumin (BSA; Sigma, St. Louis, Mo., USA) and incubated for 2 h at room temperature. Next, the plate was washed 5 times with 300 μl of washing buffer (0.05% Tween-20 in HEPES-NaCl buffer) and incubated overnight at 4°C with 100 μl mAb-ucMGP (0.69 μl/ml in 2% HNBSA; VitaK BV). Subsequently, the plate was washed 5 times with 300 μl of HEPES-Tween washing buffer. 5 μl of either serum sample or standard were diluted in 70 μl of 2% HNBSA and 20 μl of this solution were supplemented with 100 μl of tracer (biotinylated ucMGP35–54, 0.7 μg/ml). After gentle mixing, 100 μl of this solution were transferred to the microtiter plate and incubated overnight at 4°C. After 5 washing cycles with 300 μl of HEPES-Tween washing buffer, the plate was incubated with 100 μl streptavidin-peroxide (Zymed, Breda, The Netherlands) diluted 10,000-fold with 0.5% HNBSA. The plate was subsequently washed 5 times with 300 μl 0.05% HEPES-Tween washing buffer and stained with 100 μl of 3,3′,5,5′tetramethylbenzidine (KPL, Gennep, the Netherlands). The staining process was stopped by adding 50 μl of 1.0 M H2SO4, and the plate was read at 450 nm. The ucMGP concentration was calculated with the aid of a calibration curve prepared with synthetic full-length ucMGP.

goto top of outline Commercially Available MGP Assay

Serum MGP concentrations were quantified with the kit from Biomedica (Vienna, Austria). The kit is based on the competitive ELISA principle, with antibodies against non-phosphorylated MGP coated on the microtiter plate as has been described before [24].

goto top of outline Immunoprecipitation

ucMGP from plasma was purified by immunoprecipitation according to the method described by Michielsen et al. [30]. In brief, 200 μg of mAb-ucMGP were incubated for 1 hour at room temperature with 100 mg of protein A-sepharose (Pharmacia Biotech, Uppsala, Sweden). After incubation, antibodies were cross-linked with dimethyl pimelimidate (Sigma) and remaining reactive amino groups were quenched. The beads were added to a normal pool plasma sample (15 ml) and incubated overnight at 4°C. After washing of the beads with PBS, the bound fraction was collected by adding 0.1 M glycine, pH 2.7. The immunoprecipitated proteins were analyzed on SDS-PAGE. One SDS-PAGE gel was used to transfer proteins to Immobilon-P membrane for Western blot analysis using either a polyclonal antibody raised against the MGP C-terminus (designated as pAb-MGP61–79) or a monoclonal antibody raised against the MGP N-terminal peptide 3–15 (designated as mAb-MGP3–15; VitaK BV).

goto top of outline Mass Spectrometry Analysis

A second SDS-PAGE gel was used to collect the protein fraction for analysis by mass spectrometry. The MGP protein band was excised from the SDS-PAGE gel and processed on a MassPREP digestion robot (Waters). Generation of tryptic digest from the protein by in-gel digestion, MALDI-TOF analysis and subsequent database searching were performed as described previously [31]. For MS/MS analysis, 1.0 μl of the peptide mixture and 1.0 μl matrix solution (2.5 mg/ml CHCA in 50% acetonitrile/0.1% trifluoroacetic acid; TFA) were spotted on a 384-well target plate of a MALDI-TOF/TOF (4800 MALDI TOF/TOF analyzer, Applied Biosystems). Typically, 500 shots were combined for obtaining a spectrum. The generated peak list was searched with the MASCOT search engine v2.0 against the Swiss-Prot protein database for protein identification. One miss-cleavage was tolerated; carbamidomethylation was set as a fixed modification and oxidation of methionine as an optional modification. The protein charge was set at 1+. The peptide mass tolerance was set to 1 Da and the MS/MS tolerance was set at 0.2 Da. No restrictions were made on the protein mass. A protein was regarded identified when it had a significant Mascot probability score (p < 0.05).

goto top of outline Statistical Analysis

Since ucMGP concentrations were not normally distributed, non-parametric testing was used. Non-parametric correlations (Spearman’s correlation) were used to test for differences between plasma and serum ucMGP levels and to determine the correlation with age. Healthy subjects were divided into 3 groups: subjects aged <40 years, between 40 and 60 and ≥60 years. The Kruskal-Wallis test was used to test for differences between the 3 groups of healthy subjects. Mann-Whitney U tests were used to test for differences between the reference population and the different patient populations. Unless stated otherwise, all data are means of duplicate measurements and presented as means ± SD.Data processing and statistical analysis was performed using SPSS for Windows (SPSS Corp, Chicago, Ill., USA). Significance levels were based on two-tailed tests, with the alpha level set at 0.05.

 

goto top of outline Results

goto top of outline Characteristics of the ucMGP Assay

The intra-assay variability was tested for two different serum samples (ucMGP levels: 697 and 303 nM) with 21 replicates per sample in the same plate. The variation coefficients were found to be 7.2 and 10.6%, respectively. The inter-assay variability was determined using pooled serum from 9 healthy subjects which was measured in duplicate on 21 different plates from different lot numbers, on subsequent working days. The inter-assay variation coefficient was found to be 11.4%.

The lower detection limit of the assay was calculated as 3 × SD of the zero standard obtained from 21 replicates in one plate, and was found to be 21 nM. We compared the difference between citrated plasma and serum ucMGP measurements in paired samples from healthy subjects and patients, and found a good correlation (r = 0.897; p < 0.001).

The recovery after dilution was assessed by serially diluting serum from a healthy subject (mean ucMGP 869 nM) with dilution buffer (2% HNBSA) as depicted in figure 1. Further, we assessed spiking recovery and spiked serum samples depleted from MGP by immunoprecipitation (ucMGP 1.7 nM) with either synthetic full-length ucMGP or cMGP. The ratio between the expected and observed ucMGP values was 64% for full-length ucMGPand <2.5% for full-length cMGP, as measured with the ucMGP assay.

FIG01
Fig. 1. Recovery of ucMGP after dilution of serum. Serum from a healthy subject (ucMGP 869 nM) was diluted with 2% HNBSA. The theoretical (&karsi045;) as well as the observed ucMGP concentration (●) is shown. The points represent means of duplicate measurements.

To assess the assay specificity, mAb-ucMGP was tested for its cross-reaction with synthetic MGP peptides (cMGP35–54 and ucMGP35–54; fig. 2a) and synthetic full-length MGP (either cMGP or ucMGP; fig. 2b). These synthetic polypeptides were used in competitive inhibition experiments with the biotinylated ucMGP tracer (ucMGP35–54). As shown in figure 2, both ucMGP35–54 and full-length ucMGP were competitive inhibitors in the assay, whereas cMGP35–54 and full-length cMGP were not. Moreover, we tested the synthetic peptides cMGP35–49 and ucMGP35–49, in which 3 glutamate (Glu) residues are present. Both peptides did not bind to the mAb-ucMGP (data not shown). Therefore, the mAb-ucMGP recognizes only ucMGP with at least 4 Glu residues. Additionally, MGP was not detected in serum from a patient with Keutel syndrome with the ucMGP assay. It was concluded that only ucMGP is recognized by the mAb-ucMGP used in our conformation specific assay.

FIG02
Fig. 2. Reactivity of the mAb-ucMGP with synthetic MGP-derived peptides (a) and synthetic full-length MGP (b). Serum was mixed with either uncarboxylated (●) or carboxylated species (○) before testing. Points represent means ± SD (n = 12).

Identification of the product immunoprecipitated with the mAb-ucMGP was performed on Western blot analysis and showed a single band at ∼11 kDa, which reacted positively with a monoclonal antibody against the N-terminus and with a polyclonal antibody against the C-terminus of MGP (fig. 3). Analysis of the 11 kDa-band of the second SDS-PAGE gel on MALDI-TOF/TOF after in-gel digestion revealed ucMGP as protein identity in the immunoprecipitated product (table 1).

TAB01
Table 1. Identification of ucMGP by MALDI TOF/TOF

FIG03
Fig. 3. Western blot analysis of the immunoprecipitated product shows a single band at ∼11 kDa as detected with both a polyclonal antibody raised against the MGP C-terminus (pAb-MGP61–79) and a monoclonal antibody raised against the MGP N-terminus (mAb-MGP3–15).

goto top of outline Sample Preparation

To further characterize our assay, the influence of variations in the sample preparation procedure was tested using pooled serum from 20 healthy subjects (mean ucMGP 543 ± 23 nM). There was no measurable effect of centrifugation speed (1,580 and 8,000 g) during serum preparation (mean ucMGP 560 ± 10 nM and 526 ± 22 nM, respectively). Freeze-thawing up to 10 cycles did not affect the observed ucMGP concentration (mean ucMGP after 10 cycles 554 ± 38 nM).

goto top of outline Normal Range, Within-Day Variations, and Day-to-Day Variations

The healthy subjects were divided into 3 groups: subjects under the age of 40 (mean age 30 ± 6 years), those aged 40–60 years (mean age 51 ± 6 years) and those aged 60 years or more (mean age 68 ± 6 years). Baseline characteristics of the healthy subjects are shown in table 2. The normal range for ucMGP was established in the group of 54 healthy males and females under the age of 40 (reference population), with a mean serum ucMGP level of 504 ± 98 nM. The normal range of ucMGP levels, defined as the mean ± 2 × SD, was calculated to be between 308 and 699 nM; no difference was observed between males and females. The mean level of serum ucMGP in the group of subjects aged 40–60 years was 481 ± 77 nM; in the group of subjects aged 60 years or more it was 441 ± 97 nM. The mean values of these two groups were within the normal range, with tailing towards lower values (fig. 4). There was a significant difference in ucMGP levels between the three groups of healthy subjects (p = 0.02); additionally, an inverse correlation with age in the total population of healthy subjects was found (r = –0.181; p = 0.02).

TAB02
Table 2. Demographic and medical characteristics of healthy subjects

FIG04
Fig. 4. Serum ucMGP levels in the reference subjects, divided in 3 groups: subjects aged less than 40 years, aged 40–60 years and aged 60 or more. Mean ± SD ucMGP levels were 504 ± 98, 481 ± 77 and 441 ± 97 nM, respectively (depicted as horizontal bars). The difference between the 3 subpopulations was significant at p = 0.02. The normal range, defined as the mean ± 2 × SD, was established in the group of subjects aged less than 40 years (reference population) and was calculated to be between 308 and 699 nM.

The time-related variability of serum ucMGP was established in 4 healthy subjects (between 25 and 35 years old, 2 males and 2 females; mean ucMGP 578 ± 233 nM) from whom blood was taken by venipuncture at 9 time points on 1 day and subsequently on 5 different days at 9 a.m. with 1-week intervals. The within-day variation was calculated for each subject separately by expressing the SD as a percentage of the mean of the 9 time points. Coefficients of variation for each subject were found to be 6.6% (mean ucMGP 476 ± 31 nM), 15.2% (470 ± 71 nM), 10.5% (1,067 ± 112 nM), and 10.9% (485 ± 53 nM). There was no observation of a distinct circadian pattern. In a similar way, the day-to-day variation was calculated from the 6 samples obtained at weekly intervals and coefficients of variation were found to be 14.7% (513 ± 75 nM), 9.2% (449 ± 41 nM), 10.5% (1,016 ± 107 nM) and 10.7% (511 ± 55 nM).

goto top of outline Patient Samples

To evaluate the potential clinical utility of the ucMGP assay, we measured serum ucMGP in 4 different patient populations. The demographic and clinical characteristics of the patient populations are summarized in table 3. Figure 5 shows the serum ucMGP distribution in the reference (mean ucMGP 504 ± 98 nM) and angioplasty (389 ± 182 nM), aortic stenosis (312 ± 109 nM), dialysis (172 ± 82 nM) and calciphylaxis (140 ± 55 nM) population. Obviously, all 4 patient populations had significantly lower circulating ucMGP levels in comparison with the reference population (healthy subjects under the age of 40). Both the angioplasty population and aortic stenosis population showed a broader range as compared to the reference population with a strong tailing towards lower values. The ucMGP levels in hemodialysis as well as in calciphylaxis patients showed virtually no overlap with the normal range (fig. 5).

TAB03
Table 3. Demographic and medical characteristics of patients

FIG05
Fig. 5. Serum ucMGP levels in patients compared to the reference population (healthy subjects aged less than 40 years). Mean ± SD ucMGP values of angioplasty, aortic stenosis, dialysis and calciphylaxis patients were 389 ± 182, 312 ± 109, 172 ± 82, and 140 ± 55 nM, respectively (depicted as horizontal bars). Differences between the reference population and angioplasty, aortic stenosis, dialysis and calciphylaxis patients were statistically significant at p = 0.002, p < 0.001, p < 0.001 and p < 0.001, respectively.

Additionally, ucMGP levels of the patient populations were compared with the group of healthy subjects of the same age. The ucMGP level of the angioplasty population was significantly lower in comparison with the group of healthy subjects aged 40–60 years (p = 0.003). The ucMGP levels of the aortic stenosis, dialysis and calciphylaxis populations were significantly lower as compared to the group of healthy subjects aged 60 years or more (p < 0.001, p < 0.001 and p < 0.001, respectively). These results indicate that low ucMGP levels in the patient populations are not merely an effect of age.

Finally, MGP levels were measured with the commercially available MGP assay in the 4 patient populations. Only the group means of aortic stenosis and calciphylaxis patients were significantly different from the reference population (data not shown). Additionally, more than 70% of the MGP levels in the patients showed overlap with the normal range. These data do not allow individual risk assessment, since the majority of the patients cannot be discriminated from the healthy reference population.

 

goto top of outline Discussion

In this paper, we report on the development of an ELISA-based competitive assay for human ucMGP. To test the assay for its diagnostic utility, we analyzed ucMGP in 4 different patient populations. Circulating ucMGP levels were significantly lower in both groups of cardiovascular patients as well as in dialysis and calciphylaxis patients. Moreover, ucMGP levels in the dialysis and calciphylaxis patients did not overlap with those in the reference population. Clearly, serum ucMGP is a marker capable of discriminating between healthy subjects and patients with end-stage renal disease.

Dialysis patients have a 60–80% prevalence of moderate to severe vascular calcifications [32,33,34]. Calciphylaxis is by definition characterized by extensive calcifications of cutaneous arterioles usually accompanied by large vessel calcifications [35, 36]. The angioplasty and aortic stenosis patient populations each represent a different vascular pathologic etiology. Aortic stenosis patients have a high incidence of calcification but can be free of atherosclerosis, whereas patients who underwent angioplasty suffer from intimal (atherosclerotic) vascular disease. Additionally, large inter-individual variation in the extent of vascular calcification can be expected in these patient groups and could explain the overlap with the reference population.

The specificity of the assay for ucMGP was demonstrated by using synthetic MGP-related peptides;only the ucMGP peptides reacted with the mAb-ucMGP, whereas no affinity was observed for cMGP peptides. Recovery experiments using the same peptides to spike serum samples showed that only the recovery of ucMGP, and not of cMGP, was adequate. Additionally, ucMGP was not measurable in serum of a patient with Keutel syndrome – a functional knockout-human of MGP – further demonstrating the specificity of this assay for ucMGP. Finally, full-length MGP was identified by mass spectrometry as the major MGP species recognized by the mAb-ucMGP.

We hypothesized that in the presence of arterial calcification (arterial medial calcification or in atherosclerotic lesions), the circulating fraction of ucMGP may be decreased. The results obtained with our ucMGP assay show that in patient populations known for cardiovascular calcification serum ucMGP levels are significantly lower as compared to an apparently healthy reference population. The accumulation of ucMGP in atherosclerotic lesions and areas of calcification has been reported in several studies [6, 7], including our recent study using immunohistochemical techniques [5]. The balance between tissue ucMGP and circulating ucMGP in calcified arteries may be different from that in healthy ones with increased ucMGP levels in calcified tissues and consequently less ucMGP secretion in the circulation. However, the exact mechanisms including the process of MGP secretion in the circulation – remain to be elucidated. Alternative explanations for the low ucMGP levels in the selected patient populations may be lower synthesis of MGP and increased degradation of MGP before carboxylation in these patients.

A limitation of our study was the inability to measure circulating levels of cMGP. At the moment there is no assay available to measure this conformation of MGP; in the near future we aim to develop a sandwich-ELISA for cMGP as well as for ucMGP. Also, the reference population should be demonstrated to be free from disease, e.g. free from cardiovascular calcification; this would require multislice computed tomography (MSCT) or electron-beam computed tomography (EBCT) screening of all healthy volunteers. Since these imaging techniques are expensive, and also because they expose subjects to significant X-ray radiation, we have not characterized our reference population by MSCT or EBCT. However, it has been reported that the majority of subjects under the age of 40 have very low coronary artery calcium scores as measured with EBCT, with a mean Agatston score for males of 10 and for females of 9 [37]. Therefore, we defined the normal range of circulating ucMGP in the group of healthy subjects under the age of 40.

Increasingly more patient populations at risk for vascular calcification are currently being identified, including young females with systemic lupus erythematosus [38], polycystic ovary syndrome [39], and patients with alkaptonuria [40]. The inactive ucMGP fraction could have potential to serve as a noninvasive biochemical marker for cardiovascular calcification and possibly contribute to the identification of patients requiring radiological assessment of cardiovascular calcification. Obviously, diagnostic applications of ucMGP measurements would require much further study. Seen from a therapeutic perspective, vitamin K therapy has the potential of increasing the activity of MGP, and possibly reducing the development of calcification. In an animal model system, the efficacy of such therapy has recently been demonstrated [29, 41]. The effect of vitamin K therapy on circulating MGP levels remains to be determined.

The first commercially available MGP assay appears to be less suited for individual risk assessment. The cardiovascular patients as well as the patients prone for cardiovascular calcification could not be adequately separated from the reference population, since 73% of the serMGP levels in these patients were within normal range. A principal difference with our newly developed ucMGP assay is that the commercial assay does not discriminate between carboxylated and uncarboxylated MGP.

In conclusion, we have developed a robust assay for circulating ucMGP allowing the assessment of the circulating levels of uncarboxylated MGP species. It was found that different patient groups with cardiovascular disease or with a propensity to develop vascular calcification had significantly lower levels of circulating ucMGP and could be discriminated from the healthy reference population.Further studies are necessary to determine the exact correlation between ucMGP levels and the extent of vascular calcification as well as the predictive value of ucMGP measurements.

 

goto top of outline Acknowledgement

The authors thank Prof. Dr. J. Rosing for critical reading of the manuscript.


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  10. Murshed M, Schinke T, McKee MD, Karsenty G: Extracellular matrix mineralization is regulated locally; different roles of two gla-containing proteins. J Cell Biol 2004;165:625–630.
  11. Price PA, Chan WS, Jolson DM, Williamson MK: The elastic lamellae of devitalized arteries calcify when incubated in serum: evidence for a serum calcification factor. Arterioscler Thromb Vasc Biol 2006;26:1079–1085.
  12. Zebboudj AF, Imura M, Bostrom K: Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2. J Biol Chem 2002;277:4388–4394.
  13. Bostrom K, Tsao D, Shen S, Wang Y, Demer LL: Matrix GLA protein modulates differentiation induced by bone morphogenetic protein-2 in C3H10T1/2 cells. J Biol Chem 2001;276:14044–14052.
  14. Nishimoto SK, Nishimoto M: Matrix Gla protein C-terminal region binds to vitronectin: co-localization suggests binding occurs during tissue development. Matrix Biology 2005;24:353.
  15. 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;21:142–144.
  16. Keutel J, Jorgensen G, Gabriel P: A new autosomal-recessive hereditary syndrome. Multiple peripheral pulmonary stenosis, brachytelephalangia, inner-ear deafness, ossification or calcification of cartilages. Dtsch Med Wochenschr 1971;96:1676–1681.
  17. Meier M, Weng LP, Alexandrakis E, Ruschoff J, Goeckenjan G: Tracheobronchial stenosis in Keutel syndrome. Eur Respir J 2001;17:566–569.
  18. Schurgers LJ, Aebert H, Vermeer C, Bultmann B, Janzen J: Oral anticoagulant treatment: friend or foe in cardiovascular disease? Blood 2004;104:3231–3232.
  19. Koos R, Mahnken AH, Muhlenbruch G, Brandenburg V, Pflueger B, Wildberger JE, Kuhl HP: Relation of oral anticoagulation to cardiac valvular and coronary calcium assessed by multislice spiral computed tomography. Am J Cardiol 2005;96:747–749.
  20. Rosenhek R, Binder T, Porenta G, Lang I, Christ G, Schemper M, Maurer G, Baumgartner H: Predictors of outcome in severe, asymptomatic aortic stenosis. N Engl J Med 2000;343:611–617.
  21. Raggi P, Cooil B, Shaw LJ, Aboulhson J, Takasu J, Budoff M, Callister TQ: Progression of coronary calcium on serial electron beam tomographic scanning is greater in patients with future myocardial infarction. Am J Cardiol 2003;92:827–829.
  22. Raggi P, Shaw LJ, Berman DS, Callister TQ: Prognostic value of coronary artery calcium screening in subjects with and without diabetes. J Am Coll Cardiol 2004;43:1663–1669.
  23. Braam LA, Dissel P, Gijsbers BL, Spronk HM, Hamulyak K, Soute BA, Debie W, Vermeer C: Assay for human matrix gla protein in serum: potential applications in the cardiovascular field. Arterioscler Thromb Vasc Biol 2000;20:1257–1261.
  24. Schurgers LJ, Teunissen KJ, Knapen MH, Geusens P, van der Heijde D, Kwaijtaal M, van Diest R, Ketteler M, Vermeer C: Characteristics and performance of an immunosorbent assay for human matrix Gla-protein. Clin Chim Acta 2005;351:131–138.
  25. Jono S, Ikari Y, Vermeer C, Dissel P, Hasegawa K, Shioi A, Taniwaki H, Kizu A, Nishizawa Y, Saito S: Matrix Gla protein is associated with coronary artery calcification as assessed by electron-beam computed tomography. Thromb Haemost 2004;91:790–794.
  26. Moe SM, Reslerova M, Ketteler M, O’Neill K, Duan D, Koczman J, Westenfeld R, Jahnen-Dechent W, Chen NX: Role of calcification inhibitors in the pathogenesis of vascular calcification in chronic kidney disease (CKD). Kidney Int 2005;67:2295–2304.
  27. Mazzaferro S, Pasquali M, Pugliese F, Barresi G, Carbone I, Francone M, Sardella D, Taggi F: Serum levels of calcification inhibition proteins and coronary artery calcium score: comparison between transplantation and dialysis. Am J Nephrol 2007;27:75–83.
  28. Hackeng TM, Rosing J, Spronk HM, Vermeer C: Total chemical synthesis of human matrix Gla protein. Protein Sci 2001;10:864–870.
  29. Schurgers LJ, Spronk HM, Soute BA, Schiffers PM, DeMey JG, Vermeer C: Regression of warfarin-induced medial elastocalcinosis by high intake of vitamin K in rats. Blood 2007;109:2823–2831.
  30. Michielsen EC, Diris JH, Hackeng CM, Wodzig WK, Van Dieijen-Visser MP: Highly sensitive immunoprecipitation method for extracting and concentrating low-abundance proteins from human serum. Clin Chem 2005;51:222–224.
  31. Bouwman F, Suylen D, Renes J, Mariman E: Evaluation and improving the success rate of protein identification by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 2005;19:2465–2468.
  32. Raggi P, Boulay A, Chasan-Taber S, Amin N, Dillon M, Burke SK, Chertow GM: Cardiac calcification in adult hemodialysis patients: a link between end-stage renal disease and cardiovascular disease? J Am Coll Cardiol 2002;39:695–701.
  33. Goodman WG, Goldin J, Kuizon BD, Yoon C, Gales B, Sider D, Wang Y, Chung J, Emerick A, Greaser L, et al: Coronary-artery calcification in young adults with end-stage renal disease who are undergoing dialysis. N Engl J Med 2000;342:1478–1483.
  34. Block GA, Spiegel DM, Ehrlich J, Mehta R, Lindbergh J, Dreisbach A, Raggi P: Effects of sevelamer and calcium on coronary artery calcification in patients new to hemodialysis. Kidney Int 2005;68:1815–1824.
  35. Coates T, Kirkland GS, Dymock RB, Murphy BF, Brealey JK, Mathew TH, Disney AP: Cutaneous necrosis from calcific uremic arteriolopathy. Am J Kidney Dis 1998;32:384–391.
  36. Mazhar AR, Johnson RJ, Gillen D, Stivelman JC, Ryan MJ, Davis CL, Stehman-Breen CO: Risk factors and mortality associated with calciphylaxis in end-stage renal disease. Kidney Int 2001;60:324–332.
  37. Nasir K, Raggi P, Rumberger JA, Braunstein JB, Post WS, Budoff MJ, Blumenthal RS: Coronary artery calcium volume scores on electron beam tomography in 12,936 asymptomatic adults. Am J Cardiol 2004;93:1146–1149.
  38. Asanuma Y, Oeser A, Shintani AK, Turner E, Olsen N, Fazio S, Linton MF, Raggi P, Stein CM: Premature coronary-artery atherosclerosis in systemic lupus erythematosus. N Engl J Med 2003;349:2407–2415.
  39. Talbott EO, Zborowski JV, Rager JR, Boudreaux MY, Edmundowicz DA, Guzick DS: Evidence for an association between metabolic cardiovascular syndrome and coronary and aortic calcification among women with polycystic ovary syndrome. J Clin Endocrinol Metab 2004;89:5454–5461.
  40. Phornphutkul C, Introne WJ, Perry MB, Bernardini I, Murphey MD, Fitzpatrick DL, Anderson PD, Huizing M, Anikster Y, Gerber LH, et al: Natural history of alkaptonuria. N Engl J Med 2002;347:2111–2121.
  41. Spronk HM, Soute BA, Schurgers LJ, Thijssen HH, De Mey JG, Vermeer C: Tissue-specific utilization of menaquinone-4 results in the prevention of arterial calcification in warfarin-treated rats. J Vasc Res 2003;40:531–537.

 goto top of outline Author Contacts

Dr. Leon J. Schurgers
VitaK, Maastricht University
Universiteitssingel 50, PO Box 616
NL–6200 MD Maastricht (The Netherlands)
Tel. +31 43 388 1680, Fax +31 43 388 4159, E-Mail l.schurgers@bioch.unimaas.nl


 goto top of outline Article Information

Received: August 18, 2007
Accepted after revision: November 29, 2007
Published online: April 10, 2008
Number of Print Pages : 10
Number of Figures : 5, Number of Tables : 3, Number of References : 41


 goto top of outline Publication Details

Journal of Vascular Research (Incorporating 'International Journal of Microcirculation')

Vol. 45, No. 5, Year 2008 (Cover Date: August 2008)

Journal Editor: Pohl U. (Munich), Meininger G.A. (Columbia, Mo.)
ISSN: 1018–1172 (Print), eISSN: 1423–0135 (Online)

For additional information: http://www.karger.com/JVR


Copyright / Drug Dosage / Disclaimer

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Abstract

Objective: Matrix γ-carboxyglutamate (Gla) protein (MGP) is a vitamin K-dependent protein and a strong inhibitor of vascular calcification. Vitamin K deficiency leads to inactive uncarboxylated MGP (ucMGP), which accumulates at sites of arterial calcification. We hypothesized that as a result of ucMGP deposition around arterial calcification, the circulating fraction of ucMGP is decreased. Here we report on the development of an ucMGP assay and the potential diagnostic utility of monitoring serum ucMGP levels. Methods and Results: An ELISA-based assay was developed with which circulating ucMGP can be determined. Serum ucMGP levels were measured in healthy subjects (n = 165) and in four patient populations; patients who underwent angioplasty (n = 30), patients with aortic stenosis (n = 25), hemodialysis patients (n = 52), and calciphylaxis patients (n = 10). All four patient populations had significantly lower ucMGP levels. In angioplasty patients and in those with aortic stenosis, some overlap was observed with the control population. However, in the hemodialysis and calciphylaxis populations, virtually all subjects had ucMGP levels below the normal adult range. Conclusion: Serum ucMGP may be used as a biomarker to identify those at risk for developing vascular calcification. This assay may become an important tool in the diagnosis of cardiovascular calcification.



 goto top of outline Author Contacts

Dr. Leon J. Schurgers
VitaK, Maastricht University
Universiteitssingel 50, PO Box 616
NL–6200 MD Maastricht (The Netherlands)
Tel. +31 43 388 1680, Fax +31 43 388 4159, E-Mail l.schurgers@bioch.unimaas.nl


 goto top of outline Article Information

Received: August 18, 2007
Accepted after revision: November 29, 2007
Published online: April 10, 2008
Number of Print Pages : 10
Number of Figures : 5, Number of Tables : 3, Number of References : 41


 goto top of outline Publication Details

Journal of Vascular Research (Incorporating 'International Journal of Microcirculation')

Vol. 45, No. 5, Year 2008 (Cover Date: August 2008)

Journal Editor: Pohl U. (Munich), Meininger G.A. (Columbia, Mo.)
ISSN: 1018–1172 (Print), eISSN: 1423–0135 (Online)

For additional information: http://www.karger.com/JVR


Copyright / Drug Dosage

Copyright: All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher or, in the case of photocopying, direct payment of a specified fee to the Copyright Clearance Center.
Drug Dosage: The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in goverment regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any changes in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug.
Disclaimer: The statements, opinions and data contained in this publication are solely those of the individual authors and contributors and not of the publishers and the editor(s). The appearance of advertisements or/and product references in the publication is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness, quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or property resulting from any ideas, methods, instructions or products referred to in the content or advertisements.

References

  1. Vermeer C: Gamma-carboxyglutamate-containing proteins and the vitamin K-dependent carboxylase. Biochem J 1990;266:625–636.
  2. Luo G, D’Souza R, Hogue D, Karsenty G: The matrix Gla protein gene is a marker of the chondrogenesis cell lineage during mouse development. J Bone Miner Res 1995;10:325–334.
  3. Wallin R, Cain D, Sane DC: Matrix Gla protein synthesis and gamma-carboxylation in the aortic vessel wall and proliferating vascular smooth muscle cells – a cell system which resembles the system in bone cells. Thromb Haemost 1999;82:1764–1767.
  4. Berkner KL, Runge KW: The physiology of vitamin K nutriture and vitamin K-dependent protein function in atherosclerosis. J Thromb Haemost 2004;2:2118–2132.
  5. Schurgers LJ, Teunissen KJ, Knapen MH, Kwaijtaal M, van Diest R, Appels A, Reutelingsperger CP, Cleutjens JP, Vermeer C: Novel conformation-specific antibodies against matrix gamma-carboxyglutamic acid (Gla) protein: undercarboxylated matrix Gla protein as marker for vascular calcification. Arterioscler Thromb Vasc Biol 2005;25:1629–1633.
  6. Price PA, Faus SA, Williamson MK: Warfarin-induced artery calcification is accelerated by growth and vitamin D. Arterioscler Thromb Vasc Biol 2000;20:317–327.
  7. Sweatt A, Sane DC, Hutson SM, Wallin R: Matrix Gla protein (MGP) and bone morphogenetic protein-2 in aortic calcified lesions of aging rats. J Thromb Haemost 2003;1:178–185.
  8. Price PA, Faus SA, Williamson MK: Warfarin causes rapid calcification of the elastic lamellae in rat arteries and heart valves. Arterioscler Thromb Vasc Biol 1998;18:1400–1407.
  9. Luo G, Ducy P, McKee MD, Pinero GJ, Loyer E, Behringer RR, Karsenty G: Spontaneous calcification of arteries and cartilage in mice lacking matrix GLA protein. Nature 1997;386:78–81.
  10. Murshed M, Schinke T, McKee MD, Karsenty G: Extracellular matrix mineralization is regulated locally; different roles of two gla-containing proteins. J Cell Biol 2004;165:625–630.
  11. Price PA, Chan WS, Jolson DM, Williamson MK: The elastic lamellae of devitalized arteries calcify when incubated in serum: evidence for a serum calcification factor. Arterioscler Thromb Vasc Biol 2006;26:1079–1085.
  12. Zebboudj AF, Imura M, Bostrom K: Matrix GLA protein, a regulatory protein for bone morphogenetic protein-2. J Biol Chem 2002;277:4388–4394.
  13. Bostrom K, Tsao D, Shen S, Wang Y, Demer LL: Matrix GLA protein modulates differentiation induced by bone morphogenetic protein-2 in C3H10T1/2 cells. J Biol Chem 2001;276:14044–14052.
  14. Nishimoto SK, Nishimoto M: Matrix Gla protein C-terminal region binds to vitronectin: co-localization suggests binding occurs during tissue development. Matrix Biology 2005;24:353.
  15. 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;21:142–144.
  16. Keutel J, Jorgensen G, Gabriel P: A new autosomal-recessive hereditary syndrome. Multiple peripheral pulmonary stenosis, brachytelephalangia, inner-ear deafness, ossification or calcification of cartilages. Dtsch Med Wochenschr 1971;96:1676–1681.
  17. Meier M, Weng LP, Alexandrakis E, Ruschoff J, Goeckenjan G: Tracheobronchial stenosis in Keutel syndrome. Eur Respir J 2001;17:566–569.
  18. Schurgers LJ, Aebert H, Vermeer C, Bultmann B, Janzen J: Oral anticoagulant treatment: friend or foe in cardiovascular disease? Blood 2004;104:3231–3232.
  19. Koos R, Mahnken AH, Muhlenbruch G, Brandenburg V, Pflueger B, Wildberger JE, Kuhl HP: Relation of oral anticoagulation to cardiac valvular and coronary calcium assessed by multislice spiral computed tomography. Am J Cardiol 2005;96:747–749.
  20. Rosenhek R, Binder T, Porenta G, Lang I, Christ G, Schemper M, Maurer G, Baumgartner H: Predictors of outcome in severe, asymptomatic aortic stenosis. N Engl J Med 2000;343:611–617.
  21. Raggi P, Cooil B, Shaw LJ, Aboulhson J, Takasu J, Budoff M, Callister TQ: Progression of coronary calcium on serial electron beam tomographic scanning is greater in patients with future myocardial infarction. Am J Cardiol 2003;92:827–829.
  22. Raggi P, Shaw LJ, Berman DS, Callister TQ: Prognostic value of coronary artery calcium screening in subjects with and without diabetes. J Am Coll Cardiol 2004;43:1663–1669.
  23. Braam LA, Dissel P, Gijsbers BL, Spronk HM, Hamulyak K, Soute BA, Debie W, Vermeer C: Assay for human matrix gla protein in serum: potential applications in the cardiovascular field. Arterioscler Thromb Vasc Biol 2000;20:1257–1261.
  24. Schurgers LJ, Teunissen KJ, Knapen MH, Geusens P, van der Heijde D, Kwaijtaal M, van Diest R, Ketteler M, Vermeer C: Characteristics and performance of an immunosorbent assay for human matrix Gla-protein. Clin Chim Acta 2005;351:131–138.
  25. Jono S, Ikari Y, Vermeer C, Dissel P, Hasegawa K, Shioi A, Taniwaki H, Kizu A, Nishizawa Y, Saito S: Matrix Gla protein is associated with coronary artery calcification as assessed by electron-beam computed tomography. Thromb Haemost 2004;91:790–794.
  26. Moe SM, Reslerova M, Ketteler M, O’Neill K, Duan D, Koczman J, Westenfeld R, Jahnen-Dechent W, Chen NX: Role of calcification inhibitors in the pathogenesis of vascular calcification in chronic kidney disease (CKD). Kidney Int 2005;67:2295–2304.
  27. Mazzaferro S, Pasquali M, Pugliese F, Barresi G, Carbone I, Francone M, Sardella D, Taggi F: Serum levels of calcification inhibition proteins and coronary artery calcium score: comparison between transplantation and dialysis. Am J Nephrol 2007;27:75–83.
  28. Hackeng TM, Rosing J, Spronk HM, Vermeer C: Total chemical synthesis of human matrix Gla protein. Protein Sci 2001;10:864–870.
  29. Schurgers LJ, Spronk HM, Soute BA, Schiffers PM, DeMey JG, Vermeer C: Regression of warfarin-induced medial elastocalcinosis by high intake of vitamin K in rats. Blood 2007;109:2823–2831.
  30. Michielsen EC, Diris JH, Hackeng CM, Wodzig WK, Van Dieijen-Visser MP: Highly sensitive immunoprecipitation method for extracting and concentrating low-abundance proteins from human serum. Clin Chem 2005;51:222–224.
  31. Bouwman F, Suylen D, Renes J, Mariman E: Evaluation and improving the success rate of protein identification by peptide mass fingerprinting using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rapid Commun Mass Spectrom 2005;19:2465–2468.
  32. Raggi P, Boulay A, Chasan-Taber S, Amin N, Dillon M, Burke SK, Chertow GM: Cardiac calcification in adult hemodialysis patients: a link between end-stage renal disease and cardiovascular disease? J Am Coll Cardiol 2002;39:695–701.
  33. Goodman WG, Goldin J, Kuizon BD, Yoon C, Gales B, Sider D, Wang Y, Chung J, Emerick A, Greaser L, et al: Coronary-artery calcification in young adults with end-stage renal disease who are undergoing dialysis. N Engl J Med 2000;342:1478–1483.
  34. Block GA, Spiegel DM, Ehrlich J, Mehta R, Lindbergh J, Dreisbach A, Raggi P: Effects of sevelamer and calcium on coronary artery calcification in patients new to hemodialysis. Kidney Int 2005;68:1815–1824.
  35. Coates T, Kirkland GS, Dymock RB, Murphy BF, Brealey JK, Mathew TH, Disney AP: Cutaneous necrosis from calcific uremic arteriolopathy. Am J Kidney Dis 1998;32:384–391.
  36. Mazhar AR, Johnson RJ, Gillen D, Stivelman JC, Ryan MJ, Davis CL, Stehman-Breen CO: Risk factors and mortality associated with calciphylaxis in end-stage renal disease. Kidney Int 2001;60:324–332.
  37. Nasir K, Raggi P, Rumberger JA, Braunstein JB, Post WS, Budoff MJ, Blumenthal RS: Coronary artery calcium volume scores on electron beam tomography in 12,936 asymptomatic adults. Am J Cardiol 2004;93:1146–1149.
  38. Asanuma Y, Oeser A, Shintani AK, Turner E, Olsen N, Fazio S, Linton MF, Raggi P, Stein CM: Premature coronary-artery atherosclerosis in systemic lupus erythematosus. N Engl J Med 2003;349:2407–2415.
  39. Talbott EO, Zborowski JV, Rager JR, Boudreaux MY, Edmundowicz DA, Guzick DS: Evidence for an association between metabolic cardiovascular syndrome and coronary and aortic calcification among women with polycystic ovary syndrome. J Clin Endocrinol Metab 2004;89:5454–5461.
  40. Phornphutkul C, Introne WJ, Perry MB, Bernardini I, Murphey MD, Fitzpatrick DL, Anderson PD, Huizing M, Anikster Y, Gerber LH, et al: Natural history of alkaptonuria. N Engl J Med 2002;347:2111–2121.
  41. Spronk HM, Soute BA, Schurgers LJ, Thijssen HH, De Mey JG, Vermeer C: Tissue-specific utilization of menaquinone-4 results in the prevention of arterial calcification in warfarin-treated rats. J Vasc Res 2003;40:531–537.