Novel Conformation-Specific Antibodies Against Matrix γ-Carboxyglutamic Acid (Gla) Protein Undercarboxylated Matrix Gla Protein as Marker for Vascular Calcification

Cardiovascular Research Institute, Maastricht University, Maastricht, The Netherlands.
Arteriosclerosis Thrombosis and Vascular Biology (Impact Factor: 6). 09/2005; 25(8):1629-33. DOI: 10.1161/01.ATV.0000173313.46222.43
Source: PubMed


Matrix gamma-carboxyglutamic acid (Gla) protein (MGP), a vitamin K-dependent protein, is a potent in vivo inhibitor of arterial calcification. We hypothesized that low endogenous production of MGP and impaired carboxylation of MGP may contribute to the development or the progression of vascular disease.
Novel conformation-specific antibodies against MGP were used for immunohistochemistry of healthy and sclerotic arteries. In healthy arteries, MGP was mainly displayed around the elastin fibers in the tunica media. The staining colocalized with that for carboxylated MGP, whereas undercarboxylated MGP (ucMGP) was not detected. In atherosclerotic arteries, ucMGP was found in the intima, where it was associated with vesicular structures. In Mönckeberg's sclerosis of the media, ucMGP was localized around all areas of calcification. The results indicate that ucMGP is strongly associated with vascular calcification of different etiologies. In a separate study, serum MGP concentrations in a cohort of 172 subjects who had undergone percutaneous coronary intervention were significantly reduced compared with an apparently healthy population.
These data show that impaired carboxylation of MGP is associated with intimal and medial vascular calcification and suggest the essentiality of the vitamin K modification to the function of MGP as an inhibitor of ectopic calcification.

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Available from: Leon J Schurgers
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    • "The constitutive decrease in tMGP was 15%, suggesting that it can be the contribution of MGP from inferior limb veins to the total pool of circulating MGP. A study reporting low constitutive MGP expression in patients after percutaneous coronary intervention compared to a healthy control group (Schurgers et al 2005) could support our hypothesis of constitutive decrease. No significant differences regarding NT levels between controls and patients before and after surgery were found. "
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    ABSTRACT: Objectives: The main objective was to assess the interplay between circulating matrix Gla protein (MGP) - marker for vascular calcification, matrix metalloproteinase-9 (MMP-9) - marker for extracellular matrix remodeling and nitrotyrosine (NT) - marker for oxidative stress in patients with varicose veins (VV). Moreover, we wanted to investigate the behavior of these parameters before and after a stressful event (surgical removal of VV from lower limbs) and to find out if there is a contribution of MGP originating from superficial veins of the lower limb to the total pool of circulating MGP. Material and method: The pilot cohort study was accomplished on patients with VV (n=29) before and after a stressful event (surgical removal of VV from inferior limbs) and a group of age-gender matched apparently healthy volunteers (n=29). Plasma levels of tMGP, MMP-9 and NT were assayed with commercially available immunoassay kits. Results: Differences between patients with VV and age-sex matched healthy subjects were reflected only by higher levels of MMP-9 [82 (19-159) ng/mL versus 36 (2-108) ng/mL, p<0.05]; and not by circulating tMGP or NT levels. When patients before removal of VV were compared to patients after surgery, only tMGP was found significantly decreased [65 (32-97) μg/L versus 40 (17-95) μg/L, p<0.05]. We also found a correlation between tMGP and MMP-9 in patients with VV (r = 0.37, p<0.05). We did not find any correlation of NT with tMGP or MMP-9 and no significant differences in plasma NT levels in any pairwise comparisons. Conclusion: Higher circulating levels of MMP-9 could differentiate between healthy individuals and patients with chronic venous insufficiency. Consequently, oxidative stress assessed by NT did not affect circulating levels of tMGP or MMP-9 after surgical removal of VV. The constitutive decrease in plasma level of tMGP could be considered the contribution of MGP from superficial veins of the inferior limb to the total pool of circulating MGP
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    • "Undercarboxylated MGP (ucMGP) and OC (Glu-OC) are indirect markers of VK2 deficiency [34]. Specifically, the plasma level of ucMGP is an indicator of vascular calcification [35]. However, a previous study reported that a phosphorylated MGP 3-15 peptide lacking Gla residues inhibited calcification [36]. "
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    ABSTRACT: Canola oil (Can) and hydrogenated soybean oil (H2-Soy) are commonly used edible oils. However, in contrast to soybean oil (Soy), they shorten the survival of stroke-prone spontaneously hypertensive (SHRSP) rats. It has been proposed that the adverse effects of these oils on the kidney and testis are caused at least in part by dihydro-vitamin K (VK) 1 in H2-Soy and unidentified component(s) in Can. Increased intake of dihydro-VK1 is associated with decreased tissue VK2 levels and bone mineral density in rats and humans, respectively. The aim of the present study was to determine the effects of these oils on bone morphogenetic protein (BMP)-induced ectopic bone formation, which is promoted by VK2 deficiency, in relation to the role of VK in the γ-carboxylation of osteocalcin and matrix Gla protein. A crude extract of BMPs was implanted into a gap in the fascia of the femoral muscle in 5-week-old mice maintained on a Soy, Can, or H2-Soy diet. Newly formed bone volume, assessed by three-dimensional X-ray micro-computed tomography and three-dimensional reconstruction imaging for bone, was 4-fold greater in the Can and H2-Soy groups than in the Soy group. The plasma carboxylated osteocalcin (Gla-OC) and total OC (Gla-OC plus undercarboxylated osteocalcin [Glu-OC]) levels were significantly lower in the Can group than in the Soy group (p < 0.05). However, these levels did not significantly differ between the H2-Soy and Soy groups. The plasma Gla-OC/Glu-OC ratio in the Can and H2-Soy groups was significantly lower (in Can; p = 0.044) or was almost significantly lower (in H2-Soy; p = 0.053) than that in the Soy group. In conclusion, Can and H2-Soy accelerated BMP-induced bone formation in mice to a greater extent than Soy. Further research is required to evaluate whether the difference in accelerated ectopic bone formation is associated with altered levels of VK2 and VK-dependent protein(s) among the three dietary groups.
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    • "Immunohistochemistry was performed in paraffin or glycol methacrylate tissue sections to detect antigens in soft and calcified tissues, respectively [24]. Antigen retrieval of cartilage and synovial membrane tissue sections was performed by incubation with 2 mg/mL hyaluronidase (Sigma-Aldrich) and by boiling in 0.2% v/v citric acid pH 6.0, respectively. "
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    ABSTRACT: Scope: Gla-rich protein (GRP) is a vitamin K dependent protein, characterized by a high density of γ-carboxylated Glu residues, shown to accumulate in mouse and sturgeon cartilage and at sites of skin and vascular calcification in humans. Therefore, we investigated the involvement of GRP in pathological calcification in osteoarthritis (OA). Methods and results: Comparative analysis of GRP patterning at transcriptional and translational levels was performed between controls and OA patients. Using a RT-PCR strategy we unveiled two novel splice variants in human-GRP-F5 and F6-potentially characterized by the loss of full γ-carboxylation and secretion functional motifs. GRP-F1 is shown to be the predominant splice variant expressed in mouse and human adult tissues, particularly in OA cartilage, while an overexpressing human cell model points it as the major γ-carboxylated isoform. Using validated conformational antibodies detecting carboxylated or undercarboxylated GRP (c/uc GRP), we have demonstrated cGRP accumulation in controls, whereas ucGRP was the predominant form in OA-affected tissues, colocalizing at sites of ectopic calcification. Conclusion: Overall, our results indicate the predominance of GRP-F1, and a clear association of ucGRP with OA cartilage and synovial membrane. Levels of vitamin K should be further assessed in these patients to determine its potential therapeutic use as a supplement in OA treatment.
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