Vitamin K is an essential bioactive compound required for optimal body function. Vitamin K can be present in various isoforms, distinguishable by two main structures, namely, phylloquinone (K1) and menaquinones (K2). The difference in structure between K1 and K2 is seen in different absorption rates, tissue distribution, and bioavailability. Although differing in structure, both act as cofactor for the enzyme gamma-glutamylcarboxylase, encompassing both hepatic and extrahepatic activity. Only carboxylated proteins are active and promote a health profile like hemostasis. Furthermore, vitamin K2 in the form of MK-7 has been shown to be a bioactive compound in regulating osteoporosis, atherosclerosis, cancer and inflammatory diseases without risk of negative side effects or overdosing. This review is the first to highlight differences between isoforms vitamin K1 and K2 by means of source, function, and extrahepatic activity.

Rationale: Vascular calcification, the formation of calcium phosphate crystals in the vessel wall, is mediated by vascular smooth muscle cells (VSMCs). However, the underlying molecular mechanisms remain elusive precluding mechanism-based therapies. Objective: Phenotypic switching denotes a loss of contractile proteins and an increase in migration and proliferation, whereby VSMCs are termed synthetic. We examined how VSMC phenotypic switching influences vascular calcification and the possible role of the uniquely calcium-dependent ROS-forming NADPH oxidase 5 (Nox5). Methods and Results: In vitro cultures of synthetic VSMCs showed decreased expression of contractile markers CNN1, αSMA and SM22α and an increase in synthetic marker S100A4 compared to contractile VSMCs. This was associated with increased calcification of synthetic cells in response to high extracellular Ca 2+ . Phenotypic switching was accompanied by increased levels of reactive oxygen species (ROS) and Ca 2+ -dependent Nox5 in synthetic VSMCs. Nox5 itself regulated VSMC phenotype as siRNA knock-down of Nox5 increased contractile marker expression and decreased calcification, while overexpression of Nox5 decreased contractile marker expression. ROS production in synthetic VSMCs was cytosolic Ca 2+ -dependent, in line with it being mediated by Nox5. Treatment of VSMCs with Ca 2+ loaded extracellular vesicles (EVs) lead to an increase in cytosolic Ca 2+ . Inhibiting EV endocytosis with dynasore blocked the increase in cytosolic Ca 2+ and VSMC calcification. Increased ROS production resulted in increased EV release and decreased phagocytosis by VSMCs. Conclusions: We show here that contractile VSMCs are resistant to calcification and identify Nox5 as a key regulator of VSMC phenotypic switching. Additionally, we describe a new mechanism of Ca 2+ uptake via EVs and show that Ca 2+ induces ROS production in VSMCs via Nox5. ROS production is required for release of EVs, which promote calcification. Identifying molecular pathways that control Nox5 and VSMC-derived EVs provides potential targets to modulate vascular remodelling and calcification in the context of mineral imbalance.

The ageing population continues to suffer from its primary killer, cardiovascular disease (CVD). Despite recent advances in interventional medicinal and surgical therapies towards the end of the 20th century, the epidemic of cardiovascular disease has not been halted. Yet, rather than receding globally, the burden of CVD has risen to become a top cause of morbidity and mortality worldwide. Most CVD arises from thrombotic rupture of an atherosclerotic plaque, the pathologic thickening of coronary and carotid artery segments and subsequent distal ischemia in heart or brain. In fact, one-fifth of deaths are directly attributable to thrombotic rupture of a vulnerable plaque. Atherosclerotic lesion formation is caused by a concert of interactions between circulating leukocytes and platelets, interacting with the endothelial barrier, signalling into the arterial wall by the release of cytokines and extracellular vesicles (EVs). Both platelet- and cell-derived EVs represent a novel mechanism of cellular communication, particularly by the transport and transfer of cargo and by reprogramming of the recipient cell. These interactions result in phenotypic switching of vascular smooth muscle cells (VSMCs) causing migration and proliferation, and subsequent secretion of EVs. Loss of VSMCs attracts perivascular Mesenchymal Stem Cells (MSCs) from the adventitia, which are a source of VSMCs and contribute to repair after vascular injury. However, continuous stress stimuli eventually switch phenotype of cells into osteochondrogenic VSMCs facilitating vascular calcification. Although Virchow’s triad is over 100 years old, it is a reality that is accurate today. It can be briefly summarised as changes in the composition of blood (platelet EVs), alterations in the vessel wall (VSMC phenotypic switching, MSC infiltration and EV release) and disruption of blood flow (atherothrombosis). In this paper, we review the latest relevant advances in the identification of extracellular vesicle pathways as well as VSMCs and pericyte/MSC phenotypic switching, underlying vascular calcification.

Vitamin K and its essential role in coagulation (vitamin K [Koagulation]) have been well established and accepted the world over. Many countries have a Recommended Daily Intake (RDI) for vitamin K based on early research, and its necessary role in the activation of vitamin K-dependent coagulation proteins is known. In the past few decades, the role of vitamin K-dependent proteins in processes beyond coagulation has been discovered. Various isoforms of vitamin K have been identified, and vitamin K2 specifically has been highlighted for its long half-life and extrahepatic activity, whereas the dietary form vitamin K1 has a shorter half-life. In this review, we highlight the specific activity of vitamin K2 based upon proposed frameworks necessary for a bioactive substance to be recommended for an RDI. Vitamin K2 meets all these criteria and should be considered for a specific dietary recommendation intake.

Background: Vascular calcification is an active process that increases cardiovascular disease (CVD) risk. There is still no consensus on an appropriate biomarker for vascular calcification. We reasoned that the biomarker for vascular calcification is the collection of all blood components that can be sensed and integrated into a calcification response by human vascular smooth muscle cells (hVSMCs). Methods: We developed a new cell-based high-content assay, the BioHybrid assay, to measure in vitro calcification. The BioHybrid assay was compared with the o-Cresolphthalein assay and the T50 assay. Serum and plasma were derived from different cohort studies including chronic kidney disease (CKD) stages III, IV, V and VD (on dialysis), pseudoxanthoma elasticum (PXE) and other cardiovascular diseases including serum from participants with mild and extensive coronary artery calcification (CAC). hVSMCs were exposed to serum and plasma samples, and in vitro calcification was measured using AlexaFluor®-546 tagged fetuin-A as calcification sensor. Results: The BioHybrid assay measured the kinetics of calcification in contrast to the endpoint o-Cresolphthalein assay. The BioHybrid assay was more sensitive to pick up differences in calcification propensity than the T50 assay as determined by measuring control as well as pre- and post-dialysis serum samples of CKD patients. The BioHybrid response increased with CKD severity. Further, the BioHybrid assay discriminated between calcification propensity of individuals with a high CAC index and individuals with a low CAC index. Patients with PXE had an increased calcification response in the BioHybrid assay as compared to both spouse and control plasma samples. Finally, vitamin K1 supplementation showed lower in vitro calcification, reflecting changes in delta Agatston scores. Lower progression within the BioHybrid and on Agatston scores was accompanied by lower dephosphorylated-uncarboxylated matrix Gla protein levels. Conclusion: The BioHybrid assay is a novel approach to determine the vascular calcification propensity of an individual and thus may add to personalised risk assessment for CVD.