γ-Glutamyl carboxylase mutations differentially affect the biological function of vitamin K–dependent proteins

γ-Glutamyl carboxylase (GGCX) is an integral membrane protein that catalyzes posttranslational carboxylation of a number of vitamin K–dependent (VKD) proteins involved in a wide variety of physiologic processes, including blood coagulation, vascular calcification, and bone metabolism. Naturally occurring GGCX mutations are associated with multiple distinct clinical phenotypes. However, the genotype–phenotype correlation of GGCX remains elusive. Here, we systematically examined the effect of all naturally occurring GGCX mutations on the carboxylation of 3 structure–function distinct VKD proteins in a cellular environment. GGCX mutations were transiently introduced into GGCX-deficient human embryonic kidney 293 cells stably expressing chimeric coagulation factor, matrix Gla protein (MGP), or osteocalcin as VKD reporter proteins, and then the carboxylation efficiency of these reporter proteins was evaluated. Our results show that GGCX mutations differentially affect the carboxylation of these reporter proteins and the efficiency of using vitamin K as a cofactor. Carboxylation of these reporter proteins by a C-terminal truncation mutation (R704X) implies that GGCX’s C terminus plays a critical role in the binding of osteocalcin but not in the binding of coagulation factors and MGP. This has been confirmed by probing the protein–protein interaction between GGCX and its protein substrates in live cells using bimolecular fluorescence complementation and chemical cross-linking assays. Additionally, using a minigene splicing assay, we demonstrated that several GGCX missense mutations affect GGCX’s pre–messenger RNA splicing rather than altering the corresponding amino acid residues. Results from this study interpreted the correlation of GGCX’s genotype and its clinical phenotypes and clarified why vitamin K administration rectified bleeding disorders but not nonbleeding disorders

Dietary intake of menaquinone is associated with a reduced risk of coronary heart disease: the Rotterdam Study

Vitamin K-dependent proteins, including matrix Gla-protein, have been shown to inhibit vascular calcification. Activation of these proteins via carboxylation depends on the availability of vitamin K. We examined whether dietary intake of phylloquinone (vitamin K-1) and menaquinone (vitamin K-2) were related to aortic calcification and coronary heart disease (CHD) in the population-based Rotterdam Study. The analysis included 4807 subjects with dietary data and no history of myocardial infarction at baseline (1990-1993) who were followed until January 1, 2000. The risk of incident CHD, all-cause mortality, and aortic atherosclerosis was studied in tertiles of energy-adjusted vitamin K intake after adjustment for age, gender, BMI, smoking, diabetes, education, and dietary factors. The relative risk (RR) of CHD mortality was reduced in the mid and upper tertiles of dietary menaquinone compared to the lower tertile [RR = 0.73 (95% CI: 0.45, 1.17) and 0.43 (0.24, 0.77), respectively]. Intake of menaquinone was also inversely related to all-cause mortality [RR = 0.91 (0.75, 1.09) and 0.74 (0.59, 0.92), respectively] and severe aortic calcification [odds ratio of 0.71 (0.50, 1.00) and 0.48 (0.32, 0.71), respectively]. Phylloquinone intake was not related to any of the outcomes. These findings suggest tha

Magnesium but not nicotinamide prevents vascular calcification in experimental uraemia

BACKGROUND: Optimal phosphate control is an unmet need in chronic kidney disease (CKD). High serum phosphate increases calcification burden and is associated with mortality and cardiovascular disease in CKD. Nicotinamide (NA) alone or in combination with calcium-free phosphate binders might be a strategy to reduce phosphate levels and calcification and thus impact cardiovascular disease in CKD. METHODS: We studied the effect of NA alone and in combination with magnesium carbonate (MgCO3) as a potential no

Proteoglycan 4 modulates osteogenic smooth muscle cell differentiation during vascular remodeling and intimal calcification

Calcification is a prominent feature of late-stage atherosclerosis, but the mechanisms driving this process are unclear. Using a biobank of carotid endarterectomies, we recently showed that Proteoglycan 4 (PRG4) is a key molecular signature of calcified plaques, expressed in smooth muscle cell (SMC) rich regions. Here, we aimed to unravel the PRG4 role in vascular remodeling and intimal calcification. PRG4 expression in human carotid endarterectomies correlated with calcification assessed by preoperative computed tomographies. PRG4 localized to SMCs in early intimal thickening, while in advanced lesions it was found in the extracellular matrix, surrounding macro-calcifications. In experimental models, Prg4 was upregulated in SMCs from partially ligated ApoE(-/-) mice and rat carotid intimal hyperplasia, correlating with osteogenic markers and TGFb1. Furthermore, PRG4 was enriched in cells positive for chondrogenic marker SOX9 and around plaque calcifications in ApoE(-/-) mice on warfarin. In vitro, PRG4 was induced in SMCs by IFNg, TGFb1 and calcifying medium, while SMC markers were repressed under calcifying conditions. Silencing experiments showed that PRG4 expression was driven by transcription factors SMAD3 and SOX9. Functionally, the addition of recombinant human PRG4 increased ectopic SMC calcification, while arresting cell migration and proliferation. Mechanistically, it suppressed endogenous PRG4, SMAD3 and SOX9, and restored SMC markers' expression. PRG4 modulates SMC function and osteogenic phenotype during intimal remodeling and macro-calcification in response to TGFb1 signaling, SMAD3 and SOX9 activation. The effects of PRG4 on SMC phenotype and calcification suggest its role in atherosclerotic plaque stability, warranting further investigations.Vascular Surger

Vitamin K and cardiovascular complications in chronic kidney disease patients

Vitamin K, well known for its role in coagulation, encompasses 2 major subgroups: vitamin K1 is exclusively synthesized by plants, whereas vitamin K2 mostly originates from bacterial synthesis. Vitamin K serves as a cofactor for the enzyme gamma-glutamyl carboxylase, which carboxylates and thereby activates various vitamin K-dependent proteins. Several vitamin K-dependent proteins are synthesized in bone, but the role of vitamin K for bone health in chronic kidney disease patients, in particular the prevention of osteoporosis, is still not firmly established. Herein, we focus on another prominent action of vitamin K, in particular vitamin K2 (namely, the activation of matrix gamma-carboxyglutamic acid protein, the most potent inhibitor of cardiovascular calcifications). Multiple observational studies link relative vitamin K deficiency or low intake to cardiovascular calcification progress, morbidity, and mortality. Patients with advanced chronic kidney disease are particularly vitamin K deficient, in part because of dietary restrictions but possibly also due to impaired endogenous recycling of vitamin K. At the same time, this population is characterized by markedly accelerated cardiovascular calcifications and mortality. High-dose dietary supplementation with vitamin K2, in particular the most potent form, menaquinone 7, can potently reduce circulating levels of dephosphorylated uncarboxylated (i.e., inactive matrix gamma-carboxyglutamic acid protein) in patients with end-stage kidney disease. However, despite this compelling data basis, several randomized controlled trials with high-dose menaquinone 7 supplements in patients with advanced chronic kidney disease have failed to confirm cardiovascular benefits. Herein, we discuss potential reasons and solutions for this

Vitamin K and cardiovascular complications in chronic kidney disease patients

Vitamin K, well known for its role in coagulation, encompasses 2 major subgroups: vitamin K1 is exclusively synthesized by plants, whereas vitamin K2 mostly originates from bacterial synthesis. Vitamin K serves as a cofactor for the enzyme gamma-glutamyl carboxylase, which carboxylates and thereby activates various vitamin K-dependent proteins. Several vitamin K-dependent proteins are synthesized in bone, but the role of vitamin K for bone health in chronic kidney disease patients, in particular the prevention of osteoporosis, is still not firmly established. Herein, we focus on another prominent action of vitamin K, in particular vitamin K2 (namely, the activation of matrix gamma-carboxyglutamic acid protein, the most potent inhibitor of cardiovascular calcifications). Multiple observational studies link relative vitamin K deficiency or low intake to cardiovascular calcification progress, morbidity, and mortality. Patients with advanced chronic kidney disease are particularly vitamin K deficient, in part because of dietary restrictions but possibly also due to impaired endogenous recycling of vitamin K. At the same time, this population is characterized by markedly accelerated cardiovascular calcifications and mortality. High-dose dietary supplementation with vitamin K2, in particular the most potent form, menaquinone 7, can potently reduce circulating levels of dephosphorylated uncarboxylated (i.e., inactive matrix gamma-carboxyglutamic acid protein) in patients with end-stage kidney disease. However, despite this compelling data basis, several randomized controlled trials with high-dose menaquinone 7 supplements in patients with advanced chronic kidney disease have failed to confirm cardiovascular benefits. Herein, we discuss potential reasons and solutions for this

Dietary Intake of Menaquinone Is Associated with a Reduced Risk of Coronary Heart Disease: The Rotterdam Study

Vitamin K-dependent proteins, including matrix Gla-protein, have been shown to inhibit vascular calcification. Activation of these proteins via carboxylation depends on the availability of vitamin K. We examined whether dietary intake of phylloquinone (vitamin K-1) and menaquinone (vitamin K-2) were related to aortic calcification and coronary heart disease (CHD) in the population- based Rotterdam Study. The analysis included 4807 subjects with dietary data and no history of myocardial infarction at baseline (1990-1993) who were followed until January 1, 2000. The risk of incident CHD, all-cause mortality, and aortic atherosclerosis was studied in tertiles of energy-adjusted vitamin K intake after adjustment for age, gender, BMI, smoking, diabetes, education, and dietary factors. The relative risk (RR) of CHD mortality was reduced in the mid and upper tertiles of dietary menaquinone compared to the lower tertile [RR = 0.73 (95% CI: 0.45, 1.17) and 0.43 (0.24, 0.77), respectively]. Intake of menaquinone was also inversely related to all-cause mortality [RR = 0.91 (0.75, 1.09) and 0.74 (0.59, 0.92), respectively] and severe aortic calcification [odds ratio of 0.71 (0.50, 1.00) and 0.48 (0.32, 0.71), respectively]. Phylloquinone intake was not related to any of the outcomes. These findings suggest that an adequate intake of menaquinone could be important for CHD prevention

Differential Effects of Platelet Factor 4 (CXCL4) and Its Non-Allelic Variant (CXCL4L1) on Cultured Human Vascular Smooth Muscle Cells

Platelet factor 4 (CXCL4) is a chemokine abundantly stored in platelets. Upon injury and during atherosclerosis, CXCL4 is transported through the vessel wall where it modulates the function of vascular smooth muscle cells (VSMCs) by affecting proliferation, migration, gene expression and cytokine release. Variant CXCL4L1 is distinct from CXCL4 in function and expression pattern, despite a minor three-amino acid difference. Here, the effects of CXCL4 and CXCL4L1 on the phenotype and function of human VSMCs were compared in vitro. VSMCs were found to constitutively express CXCL4L1 and only exogenously added CXCL4 was internalized by VSMCs. Pre-treatment with heparin completely blocked CXCL4 uptake. A role of the putative CXCL4 receptors CXCR3 and DARC in endocytosis was excluded, but LDL receptor family members appeared to be involved in the uptake of CXCL4. Incubation of VSMCs with both CXCL4 and CXCL4L1 resulted in decreased expression of contractile marker genes and increased mRNA levels of KLF4 and NLRP3 transcription factors, yet only CXCL4 stimulated proliferation and calcification of VSMCs. In conclusion, CXCL4 and CXCL4L1 both modulate gene expression, yet only CXCL4 increases the division rate and formation of calcium-phosphate crystals in VSMCs. CXCL4 and CXCL4L1 may play distinct roles during vascular remodeling in which CXCL4 induces proliferation and calcification while endogenously expressed CXCL4L1 governs cellular homeostasis. The latter notion remains a subject for future investigation