Vascular calcification: The price to pay for anticoagulation therapy with vitamin K-antagonists

Vitamin K-antagonists (VKA) are the most widely used anti-thrombotic drugs with substantial efficacy in reducing risk of arterial and venous thrombosis. Several lines of evidence indicate, however, that VKA inhibit not only post-translational activation of vitamin K-dependent coagulation factors but also synthesis of functional extra-hepatic vitamin K-dependent proteins thereby eliciting undesired side-effects. Vascular calcification is one of the recently revealed side-effects of VKA. Vascular calcification is an actively regulated process involving vascular cells and a number of vitamin K-dependent proteins. Mechanistic understanding of vascular calcification is essential to improve VKA-based treatments of both thrombotic disorders and atherosclerosis. This review addresses vitamin K-cycle and vitamin K-dependent processes of vascular calcification that are affected by VKA. We conclude that there is a growing need for better understanding of the effects of anticoagulants on vascular calcification and atherosclerosis

Vascular calcification: The price to pay for anticoagulation therapy with vitamin K-antagonists

Vitamin K-antagonists (VKA) are the most widely used anti-thrombotic drugs with substantial efficacy in reducing risk of arterial and venous thrombosis. Several lines of evidence indicate, however, that VKA inhibit not only post-translational activation of vitamin K-dependent coagulation factors but also synthesis of functional extra-hepatic vitamin K-dependent proteins thereby eliciting undesired side-effects. Vascular calcification is one of the recently revealed side-effects of VKA. Vascular calcification is an actively regulated process involving vascular cells and a number of vitamin K-dependent proteins. Mechanistic understanding of vascular calcification is essential to improve VKA-based treatments of both thrombotic disorders and atherosclerosis. This review addresses vitamin K-cycle and vitamin K-dependent processes of vascular calcification that are affected by VKA. We conclude that there is a growing need for better understanding of the effects of anticoagulants on vascular calcification and atherosclerosis

Intra-Section Analysis of Human Coronary Arteries Reveals a Potential Role for Micro-Calcifications in Macrophage Recruitment in the Early Stage of Atherosclerosis

Vascular calcification is associated with poor cardiovascular outcome. Histochemical analysis of calcification and the expression of proteins involved in mineralization are usually based on whole section analysis, thereby often ignoring regional differences in atherosclerotic lesions. At present, limited information is available about factors involved in the initiation and progression of atherosclerosis.This study investigates the intra-section association of micro-calcifications with markers for atherosclerosis in randomly chosen section areas of human coronary arteries. Moreover, the possible causal relationship between calcifying vascular smooth muscle cells and inflammation was explored in vitro.To gain insights into the pathogenesis of atherosclerosis, we performed analysis of the distribution of micro-calcifications using a 3-MeV proton microbeam. Additionally, we performed systematic analyses of 30 to 40 regions of 12 coronary sections obtained from 6 patients including histology and immuno-histochemistry. Section areas were classified according to CD68 positivity. In vitro experiments using human vascular smooth muscle cells (hVSMCs) were performed to evaluate causal relationships between calcification and inflammation.From each section multiple areas were randomly chosen and subsequently analyzed. Depositions of calcium crystals at the micrometer scale were already observed in areas with early pre-atheroma type I lesions. Micro-calcifications were initiated at the elastica interna concomitantly with upregulation of the uncarboxylated form of matrix Gla-protein (ucMGP). Both the amount of calcium crystals and ucMGP staining increased from type I to IV atherosclerotic lesions. Osteochondrogenic markers BMP-2 and osteocalcin were only significantly increased in type IV atheroma lesions, and at this stage correlated with the degree of calcification. From atheroma area type III onwards a considerable number of CD68 positive cells were observed in combination with calcification, suggesting a pro-inflammatory effect of micro-calcifications. In vitro, invasion assays revealed chemoattractant properties of cell-culture medium of calcifying vascular smooth muscle cells towards THP-1 cells, which implies pro-inflammatory effect of calcium deposits. Additionally, calcifying hVSMCs revealed a pro-inflammatory profile as compared to non-calcifying hVSMCs.Our data indicate that calcification of VSMCs is one of the earliest events in the genesis of atherosclerosis, which strongly correlates with ucMGP staining. Our findings suggest that loss of calcification inhibitors and/or failure of inhibitory capacity is causative for the early precipitation of calcium, with concomitant increased inflammation followed by osteochondrogenic transdifferentiation of VSMCs

High-Dose Menaquinone-7 Supplementation Reduces Cardiovascular Calcification in a Murine Model of Extraosseous Calcification

Cardiovascular calcification is prevalent in the aging population and in patients with chronic kidney disease (CKD) and diabetes mellitus, giving rise to substantial morbidity and mortality. Vitamin K-dependent matrix Gla-protein (MGP) is an important inhibitor of calcification. The aim of this study was to evaluate the impact of high-dose menaquinone-7 (MK-7) supplementation (100 µg/g diet) on the development of extraosseous calcification in a murine model. Calcification was induced by 5/6 nephrectomy combined with high phosphate diet in rats. Sham operated animals served as controls. Animals received high or low MK-7 diets for 12 weeks. We assessed vital parameters, serum chemistry, creatinine clearance, and cardiac function. CKD provoked increased aortic (1.3 fold; p < 0.05) and myocardial (2.4 fold; p < 0.05) calcification in line with increased alkaline phosphatase levels (2.2 fold; p < 0.01). MK-7 supplementation inhibited cardiovascular calcification and decreased aortic alkaline phosphatase tissue concentrations. Furthermore, MK-7 supplementation increased aortic MGP messenger ribonucleic acid (mRNA) expression (10-fold; p < 0.05). CKD-induced arterial hypertension with secondary myocardial hypertrophy and increased elastic fiber breaking points in the arterial tunica media did not change with MK-7 supplementation. Our results show that high-dose MK-7 supplementation inhibits the development of cardiovascular calcification. The protective effect of MK-7 may be related to the inhibition of secondary mineralization of damaged vascular structures

<i>In vitro</i> model for chemoattractant properties of calcifying VSMCs on macrophages.

<p>The effect of calcifying VSMCs on the attraction of inflammatory cells was tested via invasion assays using PMA-stimulated THP-1 cells (macrophages). Conditioned medium of both control and calcifying VSMCs was used and calcium was added to control medium to obtain equal concentration of calcium in both conditions. Medium from calcifying VSMCs increased the invasion of macrophages significantly, indicating that VSMCs that calcify produce chemoattractants for inflammatory cells. Results were normalised to cell number at start. *P < 0.05, **P < 0.001 significance was assessed unpaired non-parametric t-test (Mann-Whitney).</p

Calcifying VSMCs <i>in Vitro</i> display a pro-inflammatory and not an osteochondrogenic phenotype.

<p>qPCR of calcifying human primary VSMCs show a significant decrease in MGP as compared to control VSMCs. No differences were found in the expression of the osteochondrogenic markers Runx2, BMP-2 and osteocalcin. The pro-inflammatory cytokines MCP1, IL1b and IFNy were significantly increased in calcifying VSMCs indicative that calcifying VSMCs can initiate local vascular inflammation and promote macrophage migration towards the vascular wall.</p

Regional differentiation of atherosclerotic lesion types.

<p>An overview of a human coronary artery section, depicting atherosclerotic stages I to IV, is shown in the left panel, with enlargements of the selected regions at the right panel. The regions of coronary lesions classified as types I, II, III, and IV, based on CD68 positivity, are shown in 1A, with corresponding regions in the calcium yield scan in 1B.</p

Model showing the potential mechanism of initiation and progression of calcification of the vascular wall.

<p>1) Contractile VSMCs in the thickened intima change phenotype towards synthetic VSMCs. Synthetic VSMCs start secreting extracellular vesicles into the extracellular environment. In case of shortage of vitamin K, a vitamin required for the conversion of ucMGP into the active form cMGP, extracellular vesicles are loaded with ucMGP which is unable to prevent nucleation of calcium-phosphate. 2) Calcifying vesicles provide the first nidus for mineralisation and microcalcifications will be formed. These microcalcifications induce an inflammatory response in VSMCs. 3) VSMCs start secreting pro-inflammatory cytokines that will attract macrophages. 4) Macrophages start fueling the inflammation process by phagocytosing mcirocalcifications and secreting pro-inflammatory cytokines. 5) Pro-inflammatory macrophages affect synthetic VSMCs which will in turn produce BMP2. Synthetic VSMCs will transdifferentiate towards osteochondrogenic VSMCs that subsequently will produce bone-forming proteins such as osteocalcin. 6) Macrocalcifications are the final result of the osteochondrogenic environment in the atherosclerotic plaque.</p