OC contains 49 amino acid residues and is synthesized and secreted by osteoblasts, odontoblasts and hypertrophic cartilage cells. The OC gene of humans is located on chromosome 1, and the earliest form of the OC protein is the OC proprotein. Subsequently, the signal peptide is removed (29). Uncarboxylated OC (uOC) is converted into carboxylated OC due to the action of GGCX and vitamin K, which acts as a coenzyme. The Glu amino acid residues are located at positions 21, 24 and 27.

OC is also termed bone Gla protein, and 1,25 (OH) ₂D₃ was reported to stimulate vitamin K2-dependent epoxidase (γ-glutamyl carboxylase) activity for OC production in cultured human osteoblasts (30). The γ-carboxylated Glu amino acid residues located in γ-carboxylated OC (cOC) have a calcium binding site that attracts calcium ions. Therefore, the compound is able to bind with calcium ions and incorporate them in the hydroxyapatite crystals that form the bone matrix (31,32), while simultaneously promoting bone mineral density (Fig. 1) (33).

The activity of osteoclasts and osteoblasts may also be regulated by cOC (34,35). OC, which originates from osteoblasts, undergoes γ-carboxylation. Following this process, a large amount is deposited in the bone matrix and a small portion (~20%) is secreted into the blood circulation. Additionally, osteotesticular protein tyrosine phosphatase (36) has been reported to have a promoting role on the degree of carboxylation in OC (Fig. 1). Previously, certain studies demonstrated that serum undercarboxylated OC may be a biomarker for osteoarthritis (OA) (37).

In the event of calcified plaques, the secretion of inflammatory factors such as interleukin-8 and monocyte chemotactic factor-1 was reduced in the blood vessels, compared with non-calcified plaques, while bone morphogenetic protein (BMP)-6, OC and other protein factors that promote bone formation were increased in calcified plaques (38). It has been suggested that calcification may exhibit a negative correlation with inflammation in patients with carotid atherosclerosis (39,40). However, the specific association and underlying mechanism of OC and atherosclerosis require further investigation.

In the prevention and treatment of osteoporosis (39), Japan's recommended intake of vitamin K2 in the form of MK-7 is 250–300 µg/day. Research has indicated that a daily dietary dose of MK-7 >100 µg/day may promote the γ-carboxylation of OC (41). During the short-term intake of MK-7, OC γ-carboxylation reduces the concentration of uOC, and long-term intake of this level of MK-7 is expected to maintain its γ-carboxylated state and promote bone metabolism, thereby enhancing bone health. In addition, human biological medicine efficacy research confirmed that MK-7 has a long period of medical efficiency and MK-7 content has significantly higher levels compared with vitamin K₁ (7–8 times higher than vitamin K₁) (41,42). MK-7 has also been reported to have a longer half-life than MK-4 (43). Among menaquinones, MK-7 is the most hydrophobic form due to its longer isoprenoid chain. The chemico-physical properties of this molecule permit its transport by plasma lipoproteins, increase its extrahepatic availability and exhibit the longest half-life (3 days) (44).

Laboratory mice are commonly employed as animal models. However, in relation to OC, certain differences exist between humans and mice. First, only a single OC gene has been reported in humans, while three different OC genes have been identified in mice. In mice, ~60% of protein sequences are conserved compared with humans (45,46). Secondly, in response to 1,25-dihydroxy vitamin D3, human OC genes are upregulated in a dose-dependent manner by 1,25-dihydroxy vitamin D3, whereas the mouse OC gene is downregulated by 1,25-dihydroxy vitamin D3 (47,48). Additionally, in the majority of species, all three vitamin-K-dependent γ-carboxyglutamic acid sites in the OC molecule are fully carboxylated. However, in humans, OC in bone and serum is incompletely carboxylated (undercarboxylated OC) (45). It has been suggested that as a direct consequence of undercarboxylation of OC, human OC concentration in bone and in the circulation are only 20% of that exhibited by other species (49).

MGP is secreted from chondrocytes, arterial medial vascular smooth muscle cells (VSMCs) (34), fibroblasts and endothelial cells. MGP is also present in a large number of tissues, including the arterial wall (50), the heart, lung, kidney and skin. It has been reported that vitamin D in bone cells may upregulate the expression of MGP (51).

The maturation of MGP involves two modification steps. Initially, dephosphorylated-uncarboxylated MGP (dp-ucMGP) is converted into undercarboxylated MGP (dp-cMGP). This process enables five glutamate residues to be converted into γ-carboxylated glutamate residues, which are termed Gla residues and provide binding sites that target apoptotic bodies, calcium ions and matrix vesicles. In the second process of modification, dp-cMGP is converted into phosphorylated-carboxylated MGP (p-cMGP). This process involves three serine residues (52,53). However, the function of these serines remains unclear (Fig. 2).

According to epidemiological research, ectopic calcification has an adverse effect on the occurrence and development of CVDs (54). It has been demonstrated via in vitro experiments that VSMC calcification is induced by elevated inorganic phosphate (Pi) uptake via a sodium-dependent phosphate cotransporter, and that such calcification is also caused by phenotypic transition from VSMCs to osteoblast-like cells and apoptotic cell death (55–61). VSMCs' osteoblastic differentiation is regulated by the upregulation of numerous osteogenic genes, including osteopontin, runt related transcription factor 2 and OC (53,57). VSMCs still affect the production of matrix vesicles. The matrix vesicles may provide a suitable microenvironment for the deposition of calcium in the vessel wall (62). Fibroblasts and mesenchymal stem cells (MSCs) in the outer membrane may also be involved in arterial calcification (63).

MGP acts as an inhibitor in the deposition and crystallisation of calcium in the blood vessel wall. Carboxylated MGP inhibits ectopic mineralisation by combining with calcium crystals, thus inhibiting their growth, and also functions through the binding and inhibition of BMP-2 (64–66). Additionally, fetuin-mineralisation complexes have been observed in animal experiments, which consist of MGP (2%), fetuin-A (80%), and calcium and phosphorus ions (18%) (Fig. 2). Fetuin-mineralisation complexes may effectively inhibit the growth, aggregation and deposition of minerals. Therefore, MGP may also affect ectopic calcification via fetuin-MGP-mineralisation complexes (67). Thus, reduced vitamin K may lead to reduced carboxylated MGP and subsequently reduced inhibition of blood vessel calcification. Therefore, the deficiency of vitamin K may increase the risk of vascular calcification.

Osteophyma, also recognised as bone hyperplasia or bone spur, refers to the formation of new bone tissue in the edge of a bone, and the calcification of cartilage and meniscus is a symptom of OA. This condition is associated with a poor quality of life in the elderly population. Currently, no suitable treatment measures are available to slow down the progression of OA (68). In epidemiological studies, low levels of circulating vitamin K have been associated with hand and knee OA cross-sections (69), with substantial knee OA progression and cartilage loss longitudinally (70).

Research has demonstrated that vitamin K deficiency in subclinical conditions may contribute to an increased risk of developing radiographic knee OA and magnetic resonance imaging-based cartilage lesions (70). In fact, cartilage cells derived from normal and OA conditions are able to produce MGP. Cartilage cells derived from OA primarily produce uncarboxylated MGP, and this form of MGP has no function. Meanwhile, cartilage cells from normal cartilage produce carboxylated MGP, the functional form of MGP, which indicates that OA may be associated with non-functional MGP (71). Therefore, we hypothesized that a deficiency in MGP carboxylation may be an important cause of OA.

Consistently low dietary consumption of vitamin K, leading to vitamin K deficiency, may lead to the inhibition of vitamin K-dependent MGP and Gas6 functions, subsequently regulating OA pathogenesis through effects on osteophytosis and cartilage destruction (72). According to a review published in 2003 (73), three VKDPs are observed in the bone: OC, MGP and protein S. In addition, certain studies have demonstrated that GRP and Gas6 are also present within the bone and cartilage (70,74). Thus, to a certain extent, the present review hypothesizes that vitamin K, particularly vitamin K2, may serve a beneficial role in the prevention of osteophytes. The specific underlying mechanism and effect require further investigation.

The circulating total-uncarboxylated MGP (t-ucMGP) concentration is 1,000 times higher compared with dp-ucMGP levels, and t-ucMGP has been suggested to consist predominantly of phosphorylated ucMGP (p-ucMGP) species. dp-ucMGP has no calcium binding group and therefore cannot be retained in blood vessels (50). In addition, a multivariate logistic analysis suggested that dp-ucMGP represents a predictor of peripheral arterial calcification independent from age, gender, previous CVD and t-ucMGP levels; and is positively associated with peripheral artery calcification (75). High levels of dp-ucMGP have also been associated with aortic calcification in patients at different stages of chronic kidney disease (CKD) (75,76).

It was previously reported that high dp-ucMGP levels were associated with an elevated risk of CVD, particularly PAD and heart failure, in patients with type 2 diabetes, while no associations between alternative MGP species and CVD risk were observed (50,77). Dalmeijer et al (77) demonstrated that low vitamin K levels may be associated with an enhanced risk of CVD. In fact, serum dp-ucMGP concentration was employed as a marker of vitamin K concentration in vascular smooth muscle and vascular calcification (78). In a previous study (79), it was revealed that that patients undergoing hemodialysis expressing the highest tertile of dp-ucMGP levels had a significantly higher calcification score than the patients in the lowest tertile. Furthermore, reduced levels of non-phosphorylated carboxylated MGP have been revealed to be associated with increased cardiovascular mortality and vascular calcifications in patients undergoing dialysis (53). Schurgers et al (75) demonstrated a positive association between dp-ucMGP levels and the calcification score in 107 patients with CKD, including 40 patients undergoing hemodialysis (75). In addition, dp-ucMGP has been associated with the severity of aortic calcification in patients with CKD (78). Furthermore, high dp-ucMGP levels have been suggested to be independently associated with below-knee arterial calcification score in patients with type 2 diabetes exhibiting normal or slightly altered kidney function, thus suggesting that estimated glomerular filtration rate remains a strong predictor of MGP levels (50).

GRP, the most recently identified member of the VKDP family, derives its name from the large number of Gla residues it contains (68). GRP was first identified in sturgeon-calcified cartilage (GRP of calcified cartilage of Adriatic sturgeon has 16 Gla residues) and is characterised by the presence of 15 putative Gla residues in man (69,80–83). Its distribution is primarily in the bone, cartilage, skin and vasculature, where it serves an important role in inhibiting vascular calcification (80,81,84,85).

GRP is considered to function as a negative regulator of osteogenic differentiation (82). GRP-F5 mice have knocked-out exons 2, while GRP-F6 mice possess knocked-out exons 3. Therefore, GRP-F5 mice exhibit total loss of γ-carboxylation action, whereas GRP-F6 mice exhibit partial loss of secretory function (86). GRP-deficient mice demonstrated no significant phenotypic alterations in the growth or calcification of skeletal structures (86). GRP may slow down the differentiation and maturation of osteoblasts and decrease the activity of alkaline phosphatase and the expression of osteogenic genes (87). In addition, GRP has been implicated in the crosstalk between inflammation and the calcification of articular tissues in OA (87). BMP-2, a protein associated with bone, was reported to be able to antagonise the expression of GRP in chondrocytes (88). Certain data has indicated that GRP may be upregulated by runt-related transcription factor 2 and osterix, subsequently inducing the differentiation of osteoblasts and the formation of nodules (89). In addition, a previously published paper demonstrated that GRP exhibited similar effects on calcification and inflammation in control and OA-derived cells (87). Therefore, GRP may have potential as a therapeutic target in OA as it has effects on calcification and inflammation processes. Comparative data indicates the co-localisation of undercarboxylated GRP at sites of ectopic calcification in cartilage and the synovial membrane in OA (86). Therefore, vitamin K insufficiency is associated with Knee OA (69,70,72). In addition, Rafael et al (86) demonstrated that the association between OA and the carboxylation deficiency of both GRP and MGP is consistent.

GRP has been reported to function as an inhibitor of calcification in the cardiovascular system (85). According to the association between GRP and BMP-2, GRP may serve a role in the regulation of VSMC differentiation into osteoblast-like cells and vascular calcification. GRP has been associated with mineralisation processes in numerous diseases that involve ectopic calcification (81,82,85,86,90). Similar to MGP, GRP acts as am inhibitor of calcification in vascular and articular tissues. The modification of GRP by γ-carboxylation is considered to be essential for its role as a mineralisation inhibitor. GRP was also reported to be involved in the mineralisation competence of VSMC-derived extracellular vesicles (44,89). Furthermore, GRP may potentially be involved in the fetuin-A-MGP calcification inhibitory system (Fig. 3).

Periostin, with a molecular weight of ~90 kDa, is a recently discovered VKDP. It contains 150 amino acid-long repeat domains and is evolutionarily conserved (91). Periostin, also termed osteoblast-specific factor 2, is an extracellular matrix (ECM) protein and a member of the fascilin-1 protein family (92). Periostin was first identified in the bones and located in the cortical periosteum (periosteum cells) and periodontal ligament. Periostin and PLF are both present in high-order vertebrates (93–96). Periostin has been reported to affect heart development (97) and cardiac remodelling (98) during the embryonic period. However, during ventricular hypertrophy and fibrosis, an increase in the expression of periostin was observed (99). Periostin is a secretory protein secreted by ECM proteins and is lipid soluble. Periostin has four repetitive fascilin domains and may interact with other ECM proteins to exhibit a variety of functions (100,101).

Periostin is primarily produced and secreted by osteoblasts and their precursor cells. However, it is also secreted by fibroblasts and is expressed in the bone and heart valves in adult mammals. Periostin has a low degree of expression in the lung and kidney (101). Periostin is an adhesion molecule that, by binding to cell surface receptors, promotes the differentiation, aggregation, adhesion and proliferation of osteoblasts. Its involvement in collagen folding is reported to be crucial for matrix assembly, which is responsible for its association with bone strength (102). Periostin has also been demonstrated to have an important effect on the development of the heart (103), and the expression of periostin in the ventriculus cordis may serve an important role in atherosclerosis (104), endocardial cushion formation and heart valve formation (97,98).

Increased expression levels of periostin may be employed as a marker of sustained high pressure of bone tissue. The level of PLF may also be used as an early indicator of adaptive bone remodelling, serving an important role in the early diagnosis and treatment of occupational musculoskeletal disorders. In adult bone, PLF is reported to be upregulated under conditions of fracture healing (105). Both periostin and PLF were expressed under conditions of mechanical overload or injury and repair of the musculoskeletal system (106). In addition, periostin markedly increased the ability of MC3T3-E1 cells to adhere to type-1 collagen or fibronectin-coated surfaces, which are established stimulators of MC3T3-E1 cell attachment (107,108).

In the hearts of mice that have experienced aortic stenosis operation (109) and cases of human heart failure (110), periostin expression levels are high. Overexpression of periostin in the heart has been reported to lead to heart dysfunction and significantly increase the risk of fibrosis (111). However, periostin-knockout mice exhibited reduced fibrosis following long-term stress overload in the heart (112). Therefore, periostin may serve an important role in cardiac fibrosis and act as a key factor in cardiac function reconstruction in heart failure following cardiac pressure overload. Periostin may exert different effects in rats and mice.

Periostin in the connective tissue is associated with mechanical force. For example, periostin was demonstrated to be expressed in the heart valve and in the glomerulus of a patient with a renal lesion (113), and was also highly upregulated following cardiac tissue injury (102). Periostin is also one of the transcription products of vascular injury. Lindner et al (114) demonstrated that periostin mRNA was detected in the smooth muscle cell inner membrane and tunica media in rats following carotid arteries sacculus injury. Both periostin and PLF are vitamin K γ-carboxylate proteins and are expressed in CVDs (106).

Periostin has four isoforms (Fig. 4), which are termed Pn-1, Pn-2, Pn-3 and Pn-4 and are distinguished by the presence or absence of exons 17 and 21 (4). Pn-1 is the full-length form, Pn-2 lacks exon 17, Pn-3 lacks exon 21 and Pn-4 lacks exons 17 and 21 (4). These isoforms of periostin were highly expressed in the hearts of rats that suffered myocardial infarction (4,115). Taniyama et al (4) indicated that the expression levels of the four forms of periostin may peak at 5–7 days post-myocardial infarction. Additionally, according to the results of in situ hybridisation in the same study, during the 5 days in the myocardial-infarcted boundary region, periostin was strongly induced, which gradually spread to the ischaemic-free wall. Furthermore (4,98), Pn-1 exhibited an inhibitory effect on the adhesion of cardiac fibroblasts and cardiac muscle cells, while Pn-2 displayed the opposite effect. Additionally, Pn-2 markedly increased the formation of blood vessels, but a similar effect was not observed for Pn-1. Pn-1 promoted cardiac fibrosis and remodelling following myocardial infarction, whereas Pn-2/4 prevented cardiac rupture in the infarcted region. According to in vitro experiments, Pn-2 and Pn-1 have the opposite effect on cell adhesion and angiogenesis, which may lead to opposing effects on cardiac remodelling (4,98).

Gas6 and plasma anticoagulant protein S have 44% homology, and the end of its amino acid sequence contains 11–12 Gla residues (116). Gas6 includes three parts, which are an amino terminal, a region that consists of repeated proteins and a carboxyl terminal. Gas6 was originally isolated from quiescent fibroblasts (117) and was also reported to be secreted from osteoblasts (118). When vitamin K is present, GGCX modifies Glu residues located in the glutamic acid residue accumulation zone and converts them to γ-Glu residues. Gas6 inhibited vascular calcification by inhibiting VSMC apoptosis (111) (Fig. 5). In addition, Gas6 may be produced by platelets and blood vessel walls, which affect thrombosis and cell formation (92).

Gas6 is highly expressed in the heart, lung, intestine, kidney, brain, spleen, ovary, testis, bone marrow, VSMCs and macrophages; however, the level of expression in the liver is low (107,108). Under pathological conditions, VSMCs differentiate into osteoblast-like cells with a secretory function, and osteoblast-like cells may synthesise and secrete osteogenesis-associated proteins. Gas6 has been demonstrated to be involved in vascular remodelling, homeostasis and atherosclerosis. There is an established association between apoptotic bodies and vascular calcifications. Gas6 has also been reported to protect endothelial cells and VSMCs against apoptosis (119,120). It has also been demonstrated that Gas6 is upregulated in human-dilated cardiomyopathy, cardiac hypertrophy of mice and in hearts treated with angiotensin II (121). Gas6 impairs the adaptation of the ventricle to chronic pressure overload by the activation of the mitogen-activated protein kinase kinase 1/2-extracellular signal-regulated kinase 1/2 signalling pathway (121). Notably, a lack of Gas6 has been reported to weaken deoxycorticosterone-induced cardiac hypertrophy and fibrosis (122).