Vascular Calcification, Vitamin K and Warfarin Therapy Possible or Plausible Connection?

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1 Basic & Clinical Pharmacology & Toxicology, 2018, 122, Doi: /bcpt MiniReview Vascular Calcification, Vitamin K and Warfarin Therapy Possible or Plausible Connection? Aino Siltari and Heikki Vapaatalo Faculty of Medicine, Pharmacology, University of Helsinki, Helsinki, Finland (Received 14 March 2017; Accepted 12 June 2017) Abstract: Atherosclerosis is a pathological process underpinning many cardiovascular diseases; it is the main cause of global mortality. Atherosclerosis is characterized by an invasion of inflammatory cells, accumulation of lipids and the formation of fatty streaks (plaques) which subsequently allow accumulation of calcium and other minerals leading to a disturbance in the vascular endothelium and its regulatory role in arterial function. Vascular calcification is a different process, stringently regulated mainly by local factors, in which osteoblast-like cells accumulate in the muscular layer of arteries ultimately taking on the physiological appearance of bone. The elevated stiffness of the arteries leads to severe vascular complications in brain, heart and kidneys. Recently, evidence from animal experiments as well as clinical and epidemiological results suggests that long-term treatment with warfarin, but not with the novel direct anticoagulants, can increase the risk or even induce vascular calcification in some individuals. Gamma-carboxylation is an enzymatic process not only needed for activation of vitamin K but also other proteins which participate in bone formation and vascular calcification. Thus, reduced expression of the vitamin K-dependent proteins which physiologically inhibit calcification of cellular matrix could be postulated to lead to vascular calcification. Published clinical data, describing at present a few thousand patients, need to be supplemented with controlled studies to confirm this interesting hypothesis. Atherosclerosis is a specific form of arteriosclerosis in which the artery wall becomes thickened as a result of invasion and accumulation of white blood cells (WBC, foam cells) and the proliferation of intimal smooth muscle cells, creating fibrofatty plaques ( fatty streaks ; fig. 1). These plaques contain both living and active WBCs (causing inflammation) and dead cells containing cholesterol and triglycerides. The remnants of the plaques contain also calcium and other materials which have become crystallized within the outermost and oldest plaques [1]. Fleckenstein [2,3], the discoverer of calcium channel blockers, has postulated that accumulation of calcium in experimental coronary plaques is crucial. He characterized two different types of coronary plaques: the calcium type evoking coronary calcinosis (as modelled by vitamin D over dosage in rats) and the cholesterol type caused by consumption of a high-cholesterol diet in rabbits. The former type can be prevented by administering calcium antagonists, while the latter type does not respond to these drugs [2,4]. Vascular calcification has been defined as a consequence of tightly regulated processes that culminate in the creation of an organized extracellular matrix deposition of osteoblast-like cells. These cells are either derived from circulating stem cells or from cells located in the vascular wall [5] (fig. 1). The osteoblast-like cells may also have differentiated from smooth Author for correspondence: Heikki Vapaatalo, Faculty of Medicine, Pharmacology, University of Helsinki, Box 63, Helsinki, Finland ( heikki.vapaatalo@helsinki.fi). muscle cells or pericytes. Several factors such as bone morphogenic proteins, oxidative stress, hyperphosphatemia (e.g. severe kidney dysfunction), parathyroid hormone and vitamin D can accelerate this transformation. In addition to stimulatory agents, lack of inhibitors of mineralization like gamma-carboxyglutamic acid, Gla protein, fetuin and osteopontin plays a role in the formation of vascular calcification. In contrast to the situation in fatty plaques, which in their early-phase damage the endothelium, vascular calcification is present mainly in the smooth muscle layer of arteries thus disturbing vascular tone and thus causing arterial stiffness [6 8]. Vitamin K is a fat-soluble vitamin similar to vitamins A and E [9]. Vitamin K exists naturally in two biologically active forms, vitamin K1 (phylloquinone) and vitamin K2 (menaquinone). All vitamin K compounds contain a naphthoquinone structure with different aliphatic side chains located in position 3. In phylloquinone, this substituent is a phytyl group, whereas in vitamin K2, it is some type of menaquinone. According to the number of isoprenoid residues in position 3, the vitamin K2 derivatives are named from MK-4 to MK-13. Vitamin K1 from food originates mainly from green vegetables or vegetable oils [10]. MK-4 is present mainly in meat, egg yolk and dairy products, MK-7 in fermented foods. Also intestinal microbes produce long-chain menaquinones MK 7 10 [11]. Compounds with vitamin K activity are absorbed from the intestine depending on their solubility [9]. In the presence of

2 20 AINO SILTARI AND HEIKKI VAPAATALO MiniReview Atherosclerotic cholesterol plaque Calcium-containing crystals Fig. 1. Simplified illustration highlighting the difference between an arteriosclerotic and a calcified artery. Fig. 2. Mechanism of warfarin-induced calcification and warfarinmetabolizing enzymes. CYP, cytochrome p450 isoenzymes; MGP, matrix Gla protein; Gas-6, growth arrest-specific protein 6; VSMC, vascular smooth muscle cells. bile acids, vitamin K1 is absorbed almost entirely via the lymphatic system. K1 is absorbed in an energy-dependent manner by a saturable process in the proximal part of small intestine. In contrast, menaquinones are absorbed by diffusion in the distal part of small intestine [9]. K1 and MKs are mainly transported to the liver. MKs are then rapidly distributed to extrahepatic tissues like bone and arterial walls. Phylloquinones are metabolized rapidly to more polar metabolites and excreted into bile and urine. To maintain a suitable vitamin K balance, daily dietary requirements should be followed [EFSA 2017: adequate intake (AI) 1 lg/kg/day for an adult, or NIH: female 90 lg and male 120 lg/day]. Phylloquinones and menaquinones are not toxic in animal studies but it is not recommended to use menadione therapeutically [9]. Vitamin K is an essential cofactor in the enzymatic carboxylation of glutamate into gamma carboxyglutamate. Vitamin K is involved in the blood coagulation process (fig. 2), playing a central role in the synthesis of many coagulation factors such as prothrombin (Factor II), and Factors VII, IX and XII in the liver as well as in the formation of circulating anticoagulants (proteins c, s and z). Vitamin K also participates in the production of several proteins involved in bone and soft tissue mineralization. Vitamin K-dependent proteins are also present in extrahepatic tissues such as bone (osteocalcin) and vascular wall (matrix Gla protein) [12]. Matrix Gla protein (MGP) has been detected in atherosclerotic plaques where it is a powerful inhibitor of calcium precipitation. For example, MGP knockout mice develop accelerated calcification of aorta and coronary arteries and die within 2 months [13]. Already in the early 1960s, it was shown that vitamin K improved the healing of bone fractures in rats. Ten years later, the presence of the first extrahepatic Gla protein, osteocalcin, was discovered and its expression is known to be activated by vitamin K. Vitamin D strengthens cross-linkages in collagen, while vitamin K-activated osteocalcin together with osteopontin improves the binding of mineralized components to collagen (See [14]). Warfarin is a 4-hydroxycoumarin derivative which was introduced in the 1940s as a rodenticide. Since the 1950s, warfarin has been widely used as an anticoagulant for prevention and treatment of thrombotic and thromboembolic diseases all around the world [15]. Warfarin decreases the hepatic synthesis and total amounts of the four vitamin K-dependent coagulation factors (fig. 2). Warfarin exists in two enantiomers (R and S) which differ in their anticoagulant potency, metabolism and elimination as well as in their interactions with numerous drugs. The commercial preparations are racemic but present in different ratios, and therefore, changing from one commercial product to another is not recommended. The oral absorption of warfarin is nearly complete and it binds avidly to plasma proteins (99% of the total plasma concentration is bound to protein) [15]. The inactive metabolites are excreted in urine and faeces. Warfarin s half-life is long, hr [15], and thus, its full effect develops slowly and lasts for a prolonged time (2 5 days) after cessation of the treatment. Furthermore, the half-lives of the coagulation proteins vary from 6 to 50 hr (Factor VII and Factor II, prothrombin, respectively). Warfarin itself is recognized as a troublesome drug, owing to extensive interindividual variation in its metabolism. S-warfarin is metabolized by a liver enzyme, CYP2C9, of which variant alleles are present in 10 20% of the Caucasian population. Additionally, dietary factors (Vitamin K) can also cause warfarin resistance, and furthermore, also some hereditary resistance has been described. The most common adverse effect is due either to inadequate dosing or too much, causing bleeding an extension of the therapeutic effect [15]. Interestingly, numerous reports originating from experimental and clinical studies as well as some observational and epidemiological data have shown that vascular calcification and ultimately also accumulation of calcium in heart

3 MiniReview WARFARIN AND VASCULAR CALCIFICATION 21 valves is possible during long-term warfarin therapy. Surprisingly, neither medical handbooks nor package leaflets have earlier mentioned this adverse effect. Only in May 2016 did the European Medical Agency (EMA) demand that the manufacturers add vascular calciphylaxis to the list of rare adverse effects. However, this phenomenon, even though it involves extraskeletal calcification, occurs in small skin vessels, not in larger arteries. Warfarin is contraindicated during pregnancy as the drug can penetrate through the placenta [15]. Vascular Calcification In recent years, several reviews on warfarin-induced vascular calcification have been published [16 19]. Our research group became also interested in this possible interaction and reported experimental [20] and clinical data on this topic [21]. So far, only a few thousand patients with the problem of vascular and/or cardiac valve calcification probably associated with or definitively caused by chronic warfarin therapy have been identified and recorded [22,23]. Old age, diabetes and especially renal insufficiency and dialysis are factors promoting extraosseal calcification. On the other hand, calcification of coronary arteries and warfarin therapy do not seem to be connected, if no previous coronary disease is evident [24]. It has been recommended [18] that warfarin therapy should be avoided if possible when there is presence of previous coronary sclerosis. In these patients, the drug may cause plaque rupture [25]. When general vascular calcification is present, the arteries become stiff thus increasing systolic and pulse pressure, pulse wave velocity with detrimental consequences like cardiac and renal insufficiency, heart infarction and cerebral haemorrhages which in turn increase cardiovascular morbidity and mortality. Furthermore, peripheral circulation is impaired [26]. In warfarin-treated haemodialysis patients, warfarin use had a hazard ratio of 1.97 for overall mortality even when confounding variables were controlled [27]. In a Chinese study [28] on chronic haemodialysis, patients with atrial fibrillation and warfarin treatment preceding haemodialysis were associated with higher risk of developing congestive heart failure (HR 1.82, 95% CI ), peripheral occlusive disease (HR 3.42, CI ) and aortic valve stenosis (HR 3.20, CI ). Mechanisms of Calcification Vascular calcification is not an irreversible, passive process; instead, it is a stringently regulated process, resembling bone formation and metabolism [6,29], and it is certainly not similar to the pathological processes causing atherosclerosis. Vitamin K is the central player in the synthesis and activation of several coagulation factors/proteins. Warfarin inhibits the hepatic reduction of vitamin K to vitamin K hydroquinone by vitamin K epoxide reductase (fig. 2). Hydroquinone is the cofactor needed for gamma-carboxylation of the glutamate residues in the formation of coagulation factors II (prothrombin), VII, IX and X. If gamma-carboxylation is defective, even though the proteins may resemble the physiological coagulation factors, they lack proteolytic activity and are not able to bind either calcium or the phospholipids needed in coagulation. It is important to be aware that the physiological coagulation proteins not only participate in haemostasis but also in many other crucial cellular processes, for example apoptosis, bone formation, vascular calcification, signalling and growth regulation [15,16,18]. In the vascular calcification process, which is distinct from atherosclerosis, smooth muscle cells in the vessel wall, endothelial and mesenchymal cells as well as hematopoietic stem cells react to inflammation, oxidative stress, metabolic and non-metabolic irritation [29]. Subsequently, smooth muscle cells begin to differentiate towards an osteogenic direction as a consequence of signalling via bone morphogenic protein (BMP) [29]. In the initial stage of calcification, small matrix vesicles are released from smooth muscle cells; these vesicles are similar to those which are liberated from chondrocytes during bone formation. It is one characteristic of this process that the vesicles do not contain inhibitors of calcification, and they accumulate matrix metalloproteinase (MMP). During the calcification of the vessel wall, several proteins normally formed in the bone, such as BMP, beta-catechin, osteopontin, osteocalcin, osteoprotegerin, matrix- gamma-carboxyglutamic acid protein (Gla), MGP, fetuin A and other factors, are present [29,30]. MGP is a small matrix protein (84 amino acids) which contains five Gla residues and a disulphide bond. MGP is expressed almost exclusively in smooth muscle cells of the vascular medial layer. It is a vitamin K-dependent protein, which inhibits calcification of cellular matrix by chelating calcium and phosphate ions. Furthermore, it is able to chelate crystals and extracellular matrix components directly via Gla or indirectly via BMP. MGP can also inhibit calcification by regulating the apoptosis of smooth muscle cells although other mechanisms have been postulated [31]. Thus, MGP is a crucial factor in inhibiting differentiation of vascular smooth muscle cells towards an osteogenic character [32 34]. The amount and activity of MGP decrease when warfarin inhibits vitamin K-dependent carboxylation processes. In an animal model (the so-called renal insufficient rat) [35] and in man as visualized by computerized tomography, it has been shown that warfarin acts on MGP expression in arteries by reducing the non-carboxylated MGP concentration in the circulation [36]. Suggested mechanisms of calcification induced by MGP are presented in fig. 3A. Growth arrest-specific protein 6 (Gas-6) is another vitamin K-dependent protein which is expressed in vascular smooth muscle cells; it inhibits their apoptosis. Small apoptotic fragments, similar to matrix vesicles, can function as a core for calcium precipitation. When warfarin inhibits carboxylation processes and activation of Gas-6, there is no longer any inhibition of apoptosis and thus calcification is initiated [16] (fig. 3B). Clinically, genetic factors of the patient and warfarin dosage are important. Sensitivity to the anticoagulant effects of warfarin are regulated by at least two genes, the subunit of vitamin K epoxide reductase complex subunit 1 (VKORC1) which is the target gene, and cytochrome P450 2C9

4 22 AINO SILTARI AND HEIKKI VAPAATALO MiniReview Fig. 3. Suggested mechanisms of vascular calcification induced by inhibition of matrix Gla protein (MGP) and growth arrest-specific protein 6 (Gas-6). (CYP2C9), which is the major enzyme involved in warfarin metabolism. Recently, a third genetic component has been described; it has been termed calumenin and it also influences warfarin sensitivity [31]. One can pose the question: Is it possible to predict the risk of vascular calcification? One potential biomarker candidate is the expression of VKORC1 as its polymorphism (VKORC1 haploid) and low enzyme activity have been associated with vascular calcification [37]. In addition, defective vitamin K- dependent decarboxylation of MGP could at least partially explain the susceptibility of arteries for calcification [31,37]. The non-carboxylated MGP in serum has also been suggested as one biological marker for the risk of vascular calcification. The CYP2C9 gene test (slow/fast metabolizers) as an indicator for warfarin metabolism might predict susceptibility to warfarin-induced vascular calcification. Possibilities to Prevent Calcification Even though extraosseal calcification is not a common adverse effect of warfarin treatment, the drug does seem to increase the risk and promote the development of vascular calcification in some individuals. No specific means for prevention are available. Evidence has been published from animal experiments for positive effects of vitamin K1 (phylloquinone) and menaquinone in warfarin-induced arterial calcification [38,39]. Epidemiological studies [40 44] on the effects of nutrition on vascular calcification suggest that vitamin K2 (menaquinone) could slightly reduce vascular calcification in general but not specifically that induced by warfarin. Therefore, the link between warfarin therapy and extraosseal calcification has been criticized [45]. Arterial calcification and osteoporosis may occur simultaneously [46 48]. For this reason, bisphosphonates have been tested in animal experiments; this therapy has been shown to prevent calcification in the vasculature without affecting bone [49]. This has been attributed to the fact that there are differences in the metabolism of vitamin K in liver (synthesis of coagulation factors) and in osteoblasts (synthesis of bone Gla protein) [50]. In other words, it should be possible to influence the effects of vitamin K in bone and other cells with similar vitamin K metabolism without affecting the coagulation process. Recently, it has been shown in in vitro as well as in animal experiments that statins have pleiotropic properties and inhibitory effects on warfarin-induced vascular calcification; these effects are not related to the cholesterol-lowering properties of these drugs [51,52], but the mechanisms are not known in detail. In our own experiment [20] in a rat model involving dietary warfarin feeding [38], neither vitamin K nor menaquinone showed any clear inhibitory effect in contrast to the outcome of a similar study [38,39]. The difference might be explained by different age of the animals, different dosing of the vitamin Ks, and especially that in our study we used rather high vitamin K1 doses to prevent bleeding which also might have antagonized warfarin effects to promote calcification. In these previous experiments, the effects of menaquinone were postulated to be mediated via the osteoprotegerin gene participating in calcification. In a clinical material [21] using laboratory data, we found correlation (adjusted on age and sex) between warfarin anticoagulation (INR values) and serum calcium (corrected with albumin). The negative correlation between these two variables, high INR (high warfarin dose) and low serum calcium (accumulation in soft tissues or increased urinary excretion) is evidence of some association between warfarin therapy and calcium metabolism in bone. Some theoretical or anecdotal observations point to other possibilities to influence extraosseal calcification by targeting matrix metalloproteinase-9 (MMP-9) and transforming growth factor beta (TGF-beta). Eicosapentaenoic acid (EPA), a fatty

5 MiniReview WARFARIN AND VASCULAR CALCIFICATION 23 acid obtained from fish oil, inhibits experimental warfarininduced vascular calcification by inhibiting MMP-9 activation, preventing the accumulation of macrophages and inhibition of osteogenic protein [37,53]. In an in vitro experiment with cultured arterial smooth muscle cells, it was demonstrated that a flavonoid, quercetin, could inhibit cellular accumulation of calcium [19]. There is also some evidence emerging from animal experiments that other compounds such as blockers of angiotensin type 1 receptors (AT1) [54] and magnesium [55] possess this property. Other Oral Anticoagulants There follows one logical question: Can new oral anticoagulants such as argatroban, bivalirudin, dabigatran (direct thrombin inhibitors) or apixaban and rivaroxaban (direct FXa inhibitors) induce vascular calcification? Their anticoagulant mechanism is not based on vitamin K metabolism [15]; therefore, it is not plausible that they would influence calcium accumulation in the arterial wall. At present, there are no reports in the literature of any arterial calcification evoked by new-generation anticoagulants and this also applies to the antithrombotic agent, acetylsalicylic acid (aspirin), which inhibits the activity of platelets. Summary and Conclusions Warfarin is often prescribed as a safe and inexpensive medicine when anticoagulation is needed; in some clinical conditions (renal insufficiency), it may be the only recommendable therapy. In certain patients for unknown reasons, there seems to be a risk of vascular calcification associated with chronic warfarin therapy. No biomarkers are available to recognize the patients at risk of suffering this rare side effect. Our proposal is that patients should consume dietary components containing vitamin K1 (phylloquinone) or vitamin K2 (menaquinones) at a rather high level, and only thereafter balance the warfarin dosage. The EFSA recommendation (2017) for daily adequate intake (AI) of vitamin K is 1 lg/kg body-weight. NIH recommends somewhat more for males, 120 lg/day, and females, 90 lg/day. Symptomatic vitamin K deficiency and impaired haemostatic control in healthy adults may take more than 2 3 weeks to develop at low phylloquinone intake (i.e. 10 lg/day). We suggest that it is advisable to clarify every patient s dietary vitamin K intake and balance it individually to the warfarin dosing. Acknowledgement We thank Ewen McDonald for checking the grammar and style of the manuscript. Source of Funding P aivikki and Sakari Sohlberg Foundation, Foundation for Clinical Chemistry, Finland (A.S.). Finska L akares allskapet, Einar and Karin Stroems Stiftelse, Finland (H.V.) 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