Therapeutic strategy for atherosclerosis based on bone-vascular axis hypothesis

Journal Pre-proof Therapeutic strategy for atherosclerosis based on bone-vascular axis hypothesis

Jeong-Min Kim, Wang-Soo Lee, Jaetaek Kim PII:

S0163-7258(19)30188-3

DOI:

https://doi.org/10.1016/j.pharmthera.2019.107436

Reference:

JPT 107436

To appear in:

Pharmacology and Therapeutics

Please cite this article as: J.-M. Kim, W.-S. Lee and J. Kim, Therapeutic strategy for atherosclerosis based on bone-vascular axis hypothesis, Pharmacology and Therapeutics(2019), https://doi.org/10.1016/j.pharmthera.2019.107436

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P&T #23554 Therapeutic strategy for atherosclerosis based on bonevascular axis hypothesis Jeong-Min Kima, Wang-Soo Leeb, Jaetaek Kim a

Department of Neurology, Chung-Ang University Hospital, Chung-Ang University College

of Medicine, Seoul, Korea Division of Cardiology, Department of Internal Medicine, Chung-Ang University Hospital,

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Chung-Ang University College of Medicine, Seoul, Korea

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Division of Endocrinology and Metabolism, Department of Internal Medicine, Chung-Ang

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University Hospital, Chung-Ang University College of Medicine, Seoul, Korea

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Corresponding authors

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Wang-Soo Lee, M.D., Ph.D.

Heart Research Institute, Cardiovascular-Arrhythmia Center, College of Medicine, Chung-

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Ang University Hospital, 102, Heukseok-ro, Dongjak-gu, Seoul, 06973, Korea

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Tel: +82-2-6299-1397 Fax: +82-2-6299-1390 E-mail: [email protected]

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and Jaetaek Kim, M.D., Ph.D.

Division of Endocrinology and Metabolism, Department of Internal Medicine, Chung-Ang University Hospital, 102 Heukseok-ro, Dongjak-gu, Seoul 06973, Korea Tel: +82-2-6299-1397 Fax: +82-2-6299-1390 E-mail: [email protected]

Number of Figures/Tables: 2/2 Number of Words: 15703

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Abstract As the world’s older population grows, the disease burden of atherosclerosis is rapidly increasing, causing significant morbidity and mortality worldwide. Despite recent improvements in the control of vascular risk factors, a significant number of patients still suffer from vascular events and the progression of atherosclerosis. Aging also results in decreased bone mineral density. Since bones are a home for hematopoietic stem cells as well as reservoirs of the minerals required for vascular integrity, it is conceivable that a novel therapeutic strategy for atherosclerosis treatment can be developed by focusing on the

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complex interplay between bones and blood vessels. The correction of mineral dyshomeostasis, disrupted bone marrow microenvironments, and triggered inflammatory cell

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production provide potential therapeutic options against the atherosclerotic process. This review highlights recent advances in our understanding of the bone-vascular link and

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discusses new insights into treatment targets for atherosclerosis.

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Keywords: Atherosclerosis; Calcification; Inflammation; Bone; Bone marrow; Mineral

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Table of Contents 1. Introduction ----------------------------------------------------------------------------------------- 5 2. Bone-vascular axis in atherosclerosis ------------------------------------------------------------6 3. Mineral homeostasis between bone and vessel wall -------------------------------------------8 4. Bone marrow microenvironment and its major cellular compartment ---------------------13

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5. Endothelial progenitor cells and circulating calcifying cells --------------------------------14

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6. Inflammatory cell mobilization from bone marrow ------------------------------------------15

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7. Inflammatory cells within atherosclerotic plaque --------------------------------------------17

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8. Two distinct calcification patterns in the vascular wall--------------------------------------19

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9. Therapeutic strategy based on bone-vascular axis -------------------------------------------20

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10. Conclusion ------------------------------------------------------------------ ---------------------27

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Abbreviations bone morphogenic protein (BMP) C-X-C motif ligand (CXCL) endothelial progenitor cells (EPC)

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F-fluorodeoxy glucose (FDG)

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F-sodium fluoride (NaF)

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fibroblast growth factor (FGF)

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genetically engineered mouse (GEM)

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hematopoietic stem cells (HSCs) interleukin (IL)

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low-density lipoprotein (LDL)

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matrix G1a protein (MGP) mesenchymal stem cells (MSC)

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osteopontin (OP)

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osteoprotegerin (OPG)

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parathyroid hormones (PTH)

positron emission tomography (PET) proprotein convertase subtilisin-kexin type 9 (PCSK9) pyrophosphate (PP)

receptor activator of nuclear factor-kB (RANK)/RANK ligand (RANKL) runt-related transcription factor 2 (RUNX2) vascular smooth muscle cells (VSMCs)

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1. Introduction Atherosclerosis is a dynamic disease process initiated by the deposition of excessively oxidized lipid materials within medium to large size arterial walls and progresses to form atherosclerotic plaques protruding into the vessel lumen (Ross, 1999; Hansson, 2005; Huynh, 2014; Koskinas et al., 2016; Park & Lee, 2019). The disruption of plaque stability by local and systemic factors can result in atherosclerotic cardiovascular or cerebrovascular event (Hansson, 2005; Elkind, 2006). Additionally, arteriosclerotic vascular wall degeneration can increase arterial stiffness and impair tissue perfusion causing target organ damage such as

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vascular dementia, cerebral small vessel disease, chronic kidney disease, and heart failure. The disease burden of atherosclerosis is rapidly increasing with the expansion of the older

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population, leading to significant morbidity and mortality worldwide (Herrington et al., 2016; GBD 2016 Stroke Collaborators, 2019). Effective strategies directed against atherosclerosis

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development and progression should be implemented to reduce healthcare costs. Fortunately, owing to the advent of anti- hypertensive and hypolipidemic therapy, mortality due to

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coronary heart disease or ischemic stroke has shown a decreasing trend, especially among the most developed countries (Herrington et al., 2016, GBD 2016 Stroke Collaborators, 2019).

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For example, the inhibition of 3-hydroxy-3-methylglutaryl-CoA reductase by statins reduces low-density lipoprotein (LDL) cholesterol levels and the risk of atherosclerotic vascular

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events. Several randomized controlled clinical trials confirmed that high- intensity statin

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treatment can effectively prevent atherosclerotic vascular events, including coronary heart disease, stroke, and vascular death, among at-risk populations (Cannon et al., 2004; LaRosa et al., 2005; Amarenco et al., 2006; Nicholls et al., 2011). In addition, ezetimibe and proprotein convertase subtilisin-kexin type 9 (PCSK9) inhibitors have been introduced into clinical use based on successful clinical trial results showing reduced LDL cholesterol levels as well as lowered cardio/cerebrovascular events beyond maximally tolerated statin (Cannon et al., 2015; Sabatine et al., 2017; Schwartz et al., 2018). Even with optimal management of traditional vascular risk factors and lipid profiles, a significant proportion of patients still suffer from vascular events with residual risk (Aday & Ridker, 2019). Up to 30 – 40% of patients are still at risk of atheroma progression, even though they are treated with the maximum tolerable dose of statin or PCSK9 inhibitor (Nicholls et al., 2011; Nicholls et al., 2016). Further, in some patients, intensive statin treatment fails to prevent cardiovascular events, such as in elderly individuals with advanced

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congestive heart failure or end-stage renal disease (Kjekshus et al., 2007; Fellström et al., 2009; Wanner et al., 2005). It has been clearly demonstrated that intensive statin treatment is associated with an increased risk of new-onset diabetes mellitus and a few unexpected instances of myopathy (Mach et al., 2018). There are also concerns among clinicians that prescribing an intensive dose of statin may result in cognitive decline in the vulnerable elderly population (Muldoon et al., 2014; Gupta et al., 2017; Mach et al., 2018), although a recent trial with the PCSK9 inhibitor did not find any relationship between cognitive decline and extremely low LDL levels, at least in the short term (Giugliano et al., 2017).

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Considering the unmet need of a treatment for atherosclerosis, a novel therapeutic strategy based on the mechanism of atherosclerosis is warranted to reduce the associated residual

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vascular risk. Therefore, development of a treatment option other than LDL lowering therapy is worth investigating. In this review, we discuss the interplay between bone and blood

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strategy based on this bone-vascular link.

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vessels in the progression of atherosclerotic plaques and suggest a potentially therapeutic

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2. Bone-vascular axis in atherosclerosis

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Aging is characterized by the progressive loss of overall physiological integrity (López-Otín et al., 2013), typically resulting in decreased bone mineral density and subsequent

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susceptibility to fractures. Several cross-sectional and longitudinal studies demonstrate an independent and inverse relationship between bone mineral density and atherosclerotic burden or arteriopathy (Lin et al., 2011; Campos-Obando et al., 2015; Hofbauer et al., 2007; Shin et al, 2018). Recent studies have disclosed the multiphasic mechanistic link between the two conditions, which cannot be explained by chronological aging itself (Demer & Tintut, 2010; Thompson & Towler, 2012). Interestingly, osteoporotic patients are more frequently associated with aortic and carotid calcification, which is a hallmark of the atherosclerotic process (Anagnostis et al., 2009; Hofbauer et al., 2007). It is easy to expect that stroke patients are likely to show decreased bone mineral density, which is associated with increased mortality, when compared to general population (Carda et al., 2009). Moreover, severe bone loss is known to be associated with silent brain infarction and cerebral white matter changes among apparently healthy elderly individuals who have no history of stroke or dementia

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(Minn et al., 2014; Myint et al., 2014). Although an exact mechanism for the paradoxical mineral dyshomeostasis between bone and blood vessels needs more investigation, researchers have found some secretory proteins that may link bone and blood vessels, such as osteoprotegerin (OPG), receptor activator of nuclear factor-kB (RANK)/RANK ligand (RANKL), the plasma fetuin- A, fibroblast growth factor (FGF)-23/Klotho, parathyroid hormones (PTH), and vitamin D (Boyle et al., 2003; Wang et al., 2005; Perwad et al., 2007; Goettsch et al., 2014; Chung et al., 2015). Since bone is known as a reservoir of hematopoietic and mesenchymal stem cells (MSC),

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which are usually located in the bone marrow throughout life, it is conceivable that the disruption of bony architecture and microenvironments can negatively affect stem and

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progenitor cell populations. The production of excessive inflammatory cells from hematopoietic organs may speed up the atherosclerotic vessel wall degeneration process,

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whereas an adequate supply of endothelial progenitor cells (EPCs) may help to maintain blood vessel wall integrity and restore target organ damage after a vascular event (Méndez-

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Ferrer et al., 2010; Morrison & Scadden, 2014). Recent studies showed that circulating microRNAs (miRNAs) or EPCs with calcification potential can affect both osteoporosis and

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atherosclerosis (Materozzi et al., 2018; Hu et al., 2017; Jia et al., 2014; Fadini et al., 2012). Circulating endothelial or mesenchymal origin progenitor cells with calcifying potential may

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be important regulators of the bone-vascular axis, although the impact on vascular integrity in

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the human body has not been fully elucidated yet (Fadini et al., 2012). MiRNAs are small, noncoding single-stranded RNAs that can inhibit the expression of specific messenger RNA by forming RNA-induced silencing complexes and binding at 3’- untranslated regions (Gennari et al., 2017; Small & Olson, 2011). Specific miRNA expression patterns have been identified in various diseases including atherosclerosis and osteoporosis. C irculating miRNAs may be another mediator between bone and vessel walls, which would have important diagnostic and therapeutic implications. Telomeres and telomerase are known to protect chromosomes against genomic instability, and their dysfunction results in rare, inherited forms of bone marrow disorders such as aplastic anemia (Calado & Young, 2009). Several studies have reported that shortened telomere length is associated with an increased risk of coronary artery disease (Willeit et al., 2010). Recently, investigators performed whole-exome sequencing to characterize clonal hematopoiesis of indeterminate potential, a condition in which there is an expanded somatic blood cell clone without hematological malignancy, and

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found that it was associated with a doubled risk of coronary heart disease in humans and with accelerated atherosclerosis in mice (Jaiswal et al., 2017). All these lines of evidence support a close mechanistic link between bone and vasculature, although the relationship might not be straightforward and may be rather heterogeneous throughout different vascular beds or different timespans of atherosclerosis (Fig. 1). The pathophysiological interaction between bone and vessel walls will be illustrated through the following scheme: the bone mineral and stem cell niche, inflammatory cell and circulating calcifying cell recruitment, and vessel wall inflammation and calcification. Furthermore, this

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multifaceted disease process indicates that a novel therapeutic strategy can be developed from

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an understanding of the complex interplay between bones and blood vessels.

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3. Mineral homeostasis between bone and vessel walls

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The paradoxical phenomenon of impaired bone mineralization accompanied by vascular calcification is especially prevalent in patients with chronic kidney disease, and possible biochemical mediators have been under active investigation in these patients (Demer & Tintut,

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2010; Nemcsik et al., 2012). Sodium thiosulfate has been investigated to reduce vascular

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calcification among the patients receiving hemodialysis, based on the therapeutic experience treating the most extreme vascular calcification phenotype, calciphylaxis or calcific uremic

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arteriolopathy (Mathews et al., 2011). Several recent studies have focused on the mediato rs such as PTH between arterial calcification and bone mineral densities (Goettsch et al., 2014). In vitro and in vivo studies with genetically engineered mouse (GEM) models have been helpful to reveal the exact pathophysiology of mineral dyshomeostasis. However, establishing causal relationships between vascular calcification and mediators affecting bone metabolism can be difficult for several reasons. First, arterial calcification observed in GEM models is mainly in the form of medial calcification of large arteries with little intimal involvement or rupture, but rupture-prone arterial calcification that causes vascular events in human atherosclerosis mainly involves intima (Doherty et al., 2003, Hofbauer et al., 2007). Therefore, direct extrapolation of cell culture or GEM study results to humans is not always possible and warrants further clinical studies (Doherty et al., 2003). Second, clinical studies sometimes provide contradictory results regarding the mediators of bone mineralization and

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vascular calcification according to the study design, focused clinical outcomes, characteristics of the included patients and measurement technique. Third, determining a causal relationship between the elevated mediator and pathologic process in human could be elusive because the elevation could either be the origin of the disease or a protective response against the disease process. We discuss here several important mediators of bone mineral homeostasis that are also suspected to play a role in atherosclerosis. The key findings and references are summarized in Table 1.

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3.1. Osteoprotegerin

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OPG is a glycoprotein that is abundantly expressed in both bones and vessel walls (Hofbauer et al., 2007). It is a member of the tumor necrosis factor receptor gene superfamily and is

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released from osteoblasts to suppress bone resorption (Hofbauer et al., 2007). It inhibits the activation of osteoclasts and promotes osteoclast apoptosis by acting as a decoy receptor for

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RANKL, thereby inhibiting RANK activation (Boyle et al., 2003, Figure 1A). OPG-deficient

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mice exhibited multiple osteoporotic fractures with severe bone loss and extensive medial calcification of aorta and renal arteries (Bucay et al., 1998). Endogenous OPG expression is localized within the smooth muscle layer of large-sized arteries, but not within veins or

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capillaries (Bucay et al., 1998). The effect of OPG expression on atherosclerosis, however, is

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complex because in vivo treatment of ApoE (-/-) mice with human OPG induced vascular fibrosis and atherosclerosis progression (Toffoli et al., 2011). A significant positive

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relationship between serum OPG concentrations and vascular calcification and mortality has also been reported among patients undergoing hemodialysis (Nitta et al., 2003; Morena et al., 2006). A recently published meta-analysis including 27,450 participants also showed that elevated circulating OPG is associated with a higher risk of future cardiovascular events, although between-study heterogeneity is relatively high (Tschiderer et al., 2018). 3.2. Pyrophosphate Pyrophosphate (PP), a ubiquitous small molecule inhibitor of mineralization in the extracellular environment, binds to calcium and other minerals to inhibit crystal growth (O’Neill et al., 2011, Fig. 1A). It is a potent inhibitor of calcium crystallization, and deficiency results in medial vascular calcification (O’Neill et al., 2011). It is degraded to phosphate by tissue non-specific alkaline phosphatase in extracellular fluids (Villa-Bellosta & Egido, 2017). Hemodialysis patients have reduced plasma PP levels, and these low levels

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were associated with increased arterial stiffness (Lomashvili et al., 2005). Since bisphosphonate is similar in chemical structure to PP, it may have a role as an inhibitor of the progression of vascular calcification (Nitta et al., 2004; Lomashvili et al., 2009). 3.3. Matrix G1a protein Matrix G1a protein (MGP) is a member of a family of mineral-binding proteins that includes several coagulation/anticoagulation factors and osteocalcin (Hofbauer et al., 2007). MGP is the most potent inhibitors of calcification by binding bone morphogenetic protein (BMP)-2

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and it requires vitamin K-dependent carboxylation for biological activation (Luo et al., 1997;

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Hermans et al., 2007). An MGP-deficient mouse model was found to exhibit extensive calcification of cartilage in proliferating chondrocytes, indicating a severe enchondral

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ossification defect at the growth plate, resulting in short stature and osteopenia (Luo et al., 1997). The elastic lamina of the arteries was also calcified, resulting in reduced vascular

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compliance and higher risk of rupture and thrombus formation (Luo et al., 1997). A vitamin

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K-dependent anticoagulant, warfarin, may worsen vascular calcification by inhibiting this pathway. While the observed impact of elevated MGP on human vasculature has been inconsistent, a recent study showed that an MGP gene polymorphism is associated with

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3.4. Fetuin-A

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al., 2018).

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increased coronary artery calcification progression (Cassidy-Bushrow et al., 2013; Barrett et

Fetuin- A is a mineral carrier protein found in bone and serum, and is a systemic inhibitor of pathological mineralization (Hofbauer et al., 2007). Therefore, its deficiency is associated with vascular calcification and increased mortality (Jahnen-Dechent et al., 2011; Wang et al., 2005). However, the relationship between fetuin- A levels and arterial stiffness is variable depending on the included population because elevated fetuin-A levels can be the result of a defensive mechanism against vascular calcification (Wang et al., 2005; Jung et al., 2010). 3.5. Osteopontin Osteopontin (OP) is a major non-collagenous bone matrix glycoprotein that binds integrins, which comprise essential receptors for osteoclast migration (Anagnostis et al., 2009). It is an acidic phosphoprotein found in mineralized tissues such as bones, and its phosphorylation promotes osteoblast differentiation (Hofbauer et al., 2007). OP exerts both pro-inflammatory

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activity after thrombin cleavage and anti- inflammatory activity inhibiting reactive oxidative species (Wolak, 2014). Whether OP actively promotes vascular calcification is not yet clear, because several studies have suggested a potentially protective vascular effect, such as attenuation of vascular calcification (Speer et al., 2002; Wolak, 2014). Histological analysis shows that it seems to be localized in calcified atherosclerotic lesions (Anagnostis et al., 2009). 3.6. Bone morphogenetic protein

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BMPs are members of the transforming growth factor β superfamily and mediate pleiotropic

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effects on various tissues (Anagnostis et al., 2009). BMPs play a crucial role in osteoblast differentiation and osteogenesis, and Smad6 is a major inhibitor of BMP signaling, mainly

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involved in the cardiovascular system (Anagnostis et al., 2009). Smad6-deficient mice show cardiac valve and outflow tract defects with increased perinatal mortality, with little effect on

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the skeletal system (Galvin et al., 2000). BMP-2 and -4 are also known to stimulate

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angiogenesis in tumor microenvironments and promote the differentiation of white adipose tissue (Kim & Choe, 2011). Recombinant human BMP-2 and BMP-7 are currently used for spinal fusion, fracture healing and dental tissue engineering, although BMP-7 treatment in a

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murine model of atherosclerosis had been shown to decrease the vascular calcification burden

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(Kim & Choe, 2011). In human calcified atheroma, BMP-2 and -4 are upregulated, along with other osteogenic proteins (Fig. 1C), and increased plasma BMP-2 levels are associated

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with atherosclerosis burden among type 2 diabetic patients (Chen et al., 2006; Dhore et al., 2001; Zhang et al., 2015). 3.7. Klotho/FGF 23

The Klotho proteins are essential components of FGF receptor complexes; they are required for the binding of FGF19, FGF21 and FGF23, which are endocrine factors regulating various metabolic processes, rather than growth factors (Kuro-O, 2019). FGF23 is secreted from osteocytes and interacts with FGF receptor-Klotho complexes in the kidneys to promote urinary phosphate excretion and to lower serum 1, 25-dihydroxyvitamin D3 levels (Shimada et al., 2004; Urakawa et al., 2006). Klotho also binds to a cell-surface receptor to interfere with insulin and insulin- like growth factor-1 signaling through the inhibition of tyrosine phosphorylation, an evolutionarily conserved mechanism for extending life span (Kurosu et al., 2005). Mice with targeted deletion of Klotho display a short life span and premature

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aging, and also exhibit infertility, skin atrophy, physical inactivity, severe osteoporosis and progressive atherosclerosis with medial calcification (Kuro-O et al., 1997). Therefore, Klotho-deficient mouse could be an ideal model to study age-related disease processes related to the disrupted bone- vascular axis (Kuro-O et al., 1997). Observation of 168 type 2 diabetes patients revealed that a decreased circulating Klotho level is a predictor of long-term macrovascular events (Pan et al., 2018). Although its role in atherosclerosis is controversial, recent observational study demonstrated that FGF23 is associated with carotid plaque burden and increased stroke risk (Shah et al., 2015; Wright et al., 2014).

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3.8. MiRNAs

MiRNAs are noncoding small RNAs consisting of 16 – 22 single-stranded nucleotides, which

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can inhibit the expression of messenger RNAs through Watson – Crick base pairing between the miRNA ‘seed region’ and sequences commonly located in the 3′ untranslated regions

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(Small & Olson 2011). Based on the base pairings, miRNAs can regulate the expression of

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multiple target genes with variable intensities, making the molecules an ideal candidate for modulation of complex biological networks such as the bone-vascular axis. A rising number of experimental reports now indicate that miRNAs contribute to every step of osteogenesis

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and bone homeostasis, from embryonic skeletal development to maintenance of adult bone

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tissue, by regulating the growth, differentiation, and activity of different cell systems inside and outside the skeleton (Gennari et al., 2017). For example, elevated miR-17-5p modulates

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osteoblastic differentiation and cell proliferation by targeting SMAD7 among patients with non-traumatic osteonecrosis (Jia et al., 2014). It is also elevated in patients with unstable coronary and cerebral artery disease (Chen et al., 2015; Kim et al., 2015). The miR-17-92 cluster is a well-known group of oncogenic miRNAs that shows pro-angiogenic functions by repressing the antiangiogenic molecules thrombospondin-1 and connective tissue growth factor (Kuhnert & Kuo, 2010). Another miRNA related to the bone and vascular system, miR-21, promotes osteoblast differentiation and mineralization, as well as inhibits osteoclastogenesis and osteoclast bone resorbing activity by targeting programmed cell death 4 protein (Gennari et al., 2017). In the cardiovascular system, miR-21 is associated with cardiac hypertrophy and fibrosis in response to pressure overload (Small & Olson, 2011). MiR-125b was the first miRNA to be associated with human coronary artery calcification, and inhibition of miR-125b result in increased runt-related transcription factor 2 (RUNX2) and Osterix expression as well as increased vascular smooth muscle cell (VSMC)

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calcification (Goettsch et al., 2011). During osteogenesis, miR-125b is known to regulate osteogenic differentiation of MSCs by targeting BMP type 1b receptor (Wang et al., 2017). The ability of miRNAs to target several disease pathways at the same time suggests potential roles in the pathophysiology of the bone-vascular axis, and understanding the mechanisms of these roles might help in development of effective therapeutic strategies (Leopold, 2014).

4. Bone marrow microenvironment and its major cellular compartment

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Besides mineral storage, bone is the primary site for stem cell maintenance and hematopoiesis. This supplies both EPCs for the maintenance of vascular integrity and

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inflammatory cells that aggravate the atherosclerotic process (Fig. 1A, B). Tremendous efforts have been made to determine the location and the constituents of the bone marrow

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niche (Méndez-Ferrer et al., 2010; Morrison & Scadden, 2014). The major structural cellular component of bone is undoubtedly osteogenic cells derived from the mesenchymal lineage,

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including osteocytes, osteoblasts, and osteoclasts. Among them, osteoblast is known to produce hematopoietic cytokines such as granulocyte colony-stimulating factor (Taichman &

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Emerson, 1994).

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The anatomical location of the HSCs niche within the bone marrow is believed to be near the sinusoidal vascular system in the endosteum with endothelial or perivascular cells (Kiel et al.,

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2005; Sugiyama et al., 2006). Histological analysis showed that most HSCs were in contact with the C-X-C chemokine ligand (CXCL) 12-positive cells surrounding the sinusoidal endothelium or endosteum (Sugiyama et al., 2006). Global deletion of CXCL12 or its receptor CXCR4 depletes HSCs from the bone marrow (Sugiyama et al., 2006). In lethally irradiated mice, purified HSCs migrate near nestin (+) MSCs in the bone marrow, while in vivo nestin (+) cell depletion significantly reduces the homing capacity of HSCs (MéndezFerrer et al., 2010). In these regards, the endothelial cells, mesenchymal lineage cells and perivascular cells near the sinusoid within bone could be functionally important constituents determining the microenvironment for bone marrow HSCs, whereas osteoblasts and osteogenic cells remain as structural determinants (Sugiyama et al., 2006; Morrison & Scadden, 2014). Other components that regulate HSC niches include sympathetic nerve fibers, non- myelinating Schwann cells, macrophages, osteoclasts, extracellular matrix and calcium

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ion (Morrison & Scadden, 2014). It is plausible that distortion of structural or functional components of bone marrow might affect the stem cell population related to vessel wall repair. Epidemiologic evidence suggests that bone mineral density is positively correlated with the number of white blood cells and red blood cells in postmenopausal women (Di Monaco et al., 2004; Kim et al., 2011). Several experimental studies have shown that the diabetic condition affects bone marrow architecture and function, thereby impairing the mobilization of immature cells into the bloodstream and regeneration potential (Fadini et al., 2014). Recent studies suggested that sleep deprivation,

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decreased physical activity and psychosocial stress could also distort the bone marrow niche and facilitate HSCs migration to the spleen to increase inflammatory cell production

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(Nahrendorf & Swirski, 2015).

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5. Endothelial progenitor cells and circulating calcifying cells The concept of EPCs, which refer to cells capable of differentiation toward mature endothelial cells for angiogenesis and vasculogenesis, was established in 1997 (Asahara et al.,

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1997), and several studies have demonstrated the impact of EPC number and function among

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patients with atherosclerotic disease (Chong et al., 2016; Fadini et al., 2012). It is also thought that some portion of EPCs can aggravate the vascular healing process after tissue

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injury (Fadini et al., 2012). Since neoangiogenesis is indispensable for new bone formation and fracture healing, circulating osteoblast-lineage cells have been identified in peripheral blood, and the fraction was reported to increase in association with pubertal growth and fracture (Eghbali-Fatourechi et al., 2005; Khosla et al., 2006). In the peripheral blood of patients with coronary artery disease, investigators found that a fraction of EPCs expressing osteogenic markers were associated with defective coronary arterial function and recurrent vascular event (Gössl et al., 2008, Fig. 1C). These cell fractions, with EPC markers including CD34, CD133 and kinase insert domain receptor, as well as calcification markers like osteocalcin or bone alkaline phosphatase, were related to the disease activity of aortic stenosis and valvular calcification (Gössl et al., 2012). Whether bone marrow-derived circulating calcifying cells are the principal components of vascular calcification, and therefore exert detrimental effect for atherosclerosis needs further investigation because there are conflicting results regarding the cellular origin of arterial calcification (Speer et al., 2009;

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Doehring et al., 2010). One study has shown that VSMCs from MGP-deficient mice gave rise to osteochondrogenic precursor cells through a genetic fate mapping strategy (Speer et al., 2009). Another study showed that CD34+/CD13+ myeloid precursor cells appeared to infiltrate the atherosclerotic plaque actively and transdifferentiated into chondrocytes-like cells (Doehring et al., 2010). Investigations from other vascular beds such as the cerebrovascular system or peripheral arteries will provide more comprehensive information regarding the biological behavior of circulating calcifying cells and their role in

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6. Inflammatory cell mobilization from bone marrow

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atherosclerosis and arterial calcification.

Inflammatory cell recruitment from hematopoietic organs such as the spleen and bone

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marrow is necessary for the initiation and progression of atherosclerosis (Gisterå & Hansson, 2017; Wang et al., 2014, Fig. 1B). Animal studies demonstrated that acute vascular injury

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elicits the production and release of inflammatory cells from hematopoietic organs, which can accelerate systemic atherosclerosis (Dutta et al., 2012). In a murine stroke model, skull bone

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marrow derived neutrophils were more likely to migrate to the adjacent brain tissue than cells from the tibia, implying that the hematopoietic structure closest to an injured tissue can

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function as a major source of inflammatory cells (Herisson et al., 2018).

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Heightened hematopoietic organ activities can be indirectly demonstrated in human subjects by applying molecular imaging technique with radioisotope such as

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F-fluorodeoxyglucose

(FDG) positron emission tomography (PET). A recent study reported that the uptake of FDG at the spleen and lumbar vertebrae was significantly increased among patients with acute coronary artery disease as compared to control subjects, and FDG uptake levels were positively correlated with systemic inflammatory markers such as high-sensitivity C-reactive protein (Kim et al., 2014). Another study demonstrated that patients with increased FDG uptake at hematopoietic organs experience more recurrent vascular events (Emami et al., 2015). Furthermore, FDG activity at hematopoietic organ is increased among the patients with stable coronary artery diseases, and its level was significantly correlated with baseline LDL cholesterol (van der Valk et al., 2017). A translational study showed that stress increases proliferation of hematopoietic progenitors, which transform toward atherosclerosis-promoting inflammatory cells in both human samples and mouse models (Heidt et al., 2014). They

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found that excess noradrenaline was released under chronic stressful conditions in atherosclerosis-prone mice, which subsequently signaled bone marrow niche cells via the β3 adrenergic receptor, thereby activating HSC proliferation and atherosclerosis progression (Heidt et al., 2014). A recent study investigated the relationship between FDG uptake levels at the brain amygdala which regulates sympathetic signals in response to fear/anxiety and those at hematopoietic organs and carotid vessel walls, and found that increased amygdala FDG uptake is independently predictive of future cardiovascular events via increased bone marrow activity and arterial inflammation (Tawakol et al., 2017). This amygdala-bone-vessel

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progression and vascular events (Tawakol et al., 2017).

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axis provides a novel insight into how emotional stress might lead to atherosclerosis

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Contrary to the coronary artery disease model, few studies have investigated bone marrow activity among patients with cerebrovascular disease or those with peripheral artery disease,

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although animal studies have demonstrated active participation of hematopoietic organs in stroke pathogenesis. In a mouse model of transient middle cerebral artery occlusion, HSCs

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increased robustly with the decrease in hematopoietic niche factors that promote stem cell quiescence (Courties et al., 2015). In mice with a genetic deficiency of the β3 adrenergic

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receptor, the proliferation of the hematopoietic compartment was not observed, suggesting ischemic stroke activated HSCs within the bone marrow via increased sympathetic tone

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(Courties et al., 2015). Since the spleen is the largest reservoir of lymphocytes, splenectomy

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can modulate the acute inflammatory response following acute stroke, but its effect on infarction size and neurological recovery are variable depending on the animal strain and timing of surgery (Zierath et al., 2017). Although bone marrow activity after stroke has not yet been investigated, a recent study showed that patients with asymptomatic intracranial atherosclerosis detected from computed tomography angiography had decreased FDG uptake at lumbar vertebrae and a non-significant increase at the spleen, which is contrary to the observations from coronary artery studies (Kim et al., 2019a, Fig. 2A). Whether this contradictory result between cerebral and coronary artery disease suggest differential roles of hematopoietic organs in different vascular beds or results from different demographic and clinical characteristics of included subjects requires longitudinal follow- up and additional studies. Nevertheless, FDG uptake levels at vertebral bodies may not solely depend upon inflammatory cell producing activity, since diverse cell populations reside within vertebrae and multiple cellular components including osteoblasts/osteoclasts, MSCs, HSCs and other

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supporting cells for a bone marrow niche (Méndez-Ferrer et al., 2010).

7. Inflammatory cells within atherosclerotic plaque When atheromatous plaque starts to form at a region of disturbed blood flow with low shear stress, its progression requires a cellular supply, involving recruitment from distant organs such as bone marrow and spleen, and a phenotype switch/proliferation of resident progenitor cells and VSMCs. Trans-differentiation and migration of VSMCs are facilitated by

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lipoprotein accumulation, endothelial activation, and inflammatory responses (Tabas et al., 2015). This process can result in diverse cellular phenotypes, including smooth muscle cells,

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macrophages, and osteogenic progenitors, thereby accelerating atheroma formation and growth (Lao et al., 2015; Bentzon et al., 2006). Interestingly, some hematopoietic driven

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monocytes or even endothelial cells can transform into a smooth muscle cell- like phenotype, which makes it difficult to ascertain the exact cellular origin of an individual cell (Tabas et al.,

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2015; Lao et al., 2015). This proliferative capacity can be enhanced by various signals derived from the atheroma microenvironment, which in turn increases the atheroma volume,

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complexity, and overall vulnerability (Lao et al., 2015). The dynamic macrophage phenotype switch is also important in determination of aggravation or resolution of inflammation within

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atheroma (Nakahara et al., 2017; Lavine et al., 2018).

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The application of molecular imaging can quantify diverse pathological steps within atheroma by applying several radioligands (Tarkin et al., 2014, Fig. 2B). FDG was the first and the most commonly used radioisotope in atherosclerosis research, and it reflects the activity of plaque macrophages because macrophages infiltrated within the atheroma are metabolically active and require more glucose than other supporting cells (Tarkin et al., 2014). Recently, many other radioligands have been applied to detect plaque vulnerability: 68Ga[1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid]‑ d-Phe(1),Tyr(3)-octreotate for somatostatin receptors in macrophage, calcification, and

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F-sodium fluoride (NaF) for hydroxyapatite in

F-fluoromisonidazole for hypoxia (Tarkin et al., 2014). A recent study

compared FDG and NaF ligands for the detection of culprit plaque among patients with coronary atherosclerosis and reported that NaF is superior to FDG (Joshi et al., 2014). The same study group also showed that NaF ligands could detect disease activity in aortic valve stenosis (Dweck et al., 2012). These results stem from the background FDG uptake from

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normal myocardial tissue and the increased NaF uptake in microcalcification which increases overall instability of coronary atheroma (Joshi et al., 2014). A mechanistic study demonstrated that NaF binds to not only normal bony structures but also microcalcification within the necrotic core of carotid atheroma (Irkle et al., 2015). When these two radioligands had been used to investigate carotid atherosclerosis among stroke patients, the authors reported that both radiotracers were prevalently found in carotid atheroma, although the FDG ligand seemed to be superior to NaF (Kim et al., 2019b; Kim et al., 2019c, Fig. 2B). The different results between coronary and carotid vasculature seemed to be due to the different

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background FDG uptake and the pathological role affecting atheroma vulnerability. Since the

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heart is the most mobile organ in our body and pumps several thousand times per hour, mechanistic stress by calcified segments within a vessel wall may be important factor

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increasing atheroma vulnerability. These differences could also be due to different amounts of calcification burden and the role of plaque vulnerability among diverse ethnicities (Vesey et

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al., 2017). More studies are warranted to determine the optimal radioligand to detect culprit

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atheromatous plaque in different vascular beds.

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8. Two distinct calcification patterns in the vascular wall

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Vascular wall calcification is an active process in the progression of atherosclerosis that

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develops as a defensive mechanism against atheroma rupture. This may prevent necrotic material from entering the blood vessel lumen, but increases arterial stiffness thereby decreasing end-organ perfusion (Fig. 1C). While the blood vessel wall has three layers, intima, media, and adventitia, the calcification process mainly occurs in intima and media of the arterial wall depending on the pathophysiology, and involves crystallization of calcium/phosphate in the form of hydroxyapatite in the extracellular matrix (Virmani et al., 2000; Wolisi & Moe, 2005; Ho & Shanahan 2016). In healthy vessel walls, VSMCs and resident macrophages contain calcification inhibitors, including MGP and fetuin-A, which minimize vascular calcification and facilitate rapid phagocytosis of apoptotic particles (Reynolds et al., 2005; Proudfoot et al., 2002, Fig. 1A). Other potent anti-calcification factors include PP, OP, Klotho, and OPG (Nakahara et al., 2017, Fig. 1A). Intimal calcification is usually due to atherosclerotic inflammatory process, which resembles endochondral bone formation in long bones and aggravates plaque instability (Nakahara et al.,

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2017). Oxysterol from oxidized LDL cholesterol and hydrogen pero xide upregulate osteogenic BMPs and RUNX2 also known as core-binding factor subunit alpha-1 expression, which results in an osteochondrogenic phenotype switch of the VSMCs (Richardson et al., 2007;

Byon

et

al.,

2008).

In

line

with

the

bone- vascular

axis

hypothesis,

hypercholesterolemia and oxidized cholesterol suppress bone formation in atherosclerosissusceptible mice (Parhami et al., 1997; Parhami et al., 2001). Medial calcification, which is prevalent among patients with type 2 diabetes and chronic kidney diseases, occurs in the form of mineralization along mural elastin fibers resembling

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intramembranous bone formation. This calcification impairs the vessel mechanics and Windkessel physiology necessary for smooth tissue perfusion, rather than causing atheroma

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rupture or vessel lumen stenosis (O'Rourke & Hashimoto, 2007; Nakahara et al., 2017). The activation of Msh homeobox 1 & 2, along with OP and BMP, contributes to the pro-

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osteogenic milieu of tunica media (Towler et al., 1998; Cheng et al., 2014, Fig. 1C). These signals recruit vascular mesenchymal cell populations with pericyte characteristics and

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calcification potential, such as circulating vascular cells, adventitial myofibroblasts and valve

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interstitial cells (Boström et al., 2011; Fadini et al., 2011).

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9. Therapeutic strategies based on the bone-vascular axis

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Since bone mineral loss and vascular calcification share common pathophysiologic machinery and risk factors, it is conceivable that an effective therapeutic strategy for one disease may be also effective to modulate another pathologic condition. Indeed, several lines of evidence showed that statin treatment not only reduce s the progression of atherosclerosis, but also inhibits the development of osteoporosis and restores bone mineral density (Lin et al., 2018). In this section we summarized the impact of anti-osteoporotic therapeutics on the vascular system. We also mentioned contradictory clinical trial results from two recently studied anti- inflammatory drugs for the atherosclerotic cardiovascular disease as the findings might be due to the differential effects of the drugs on the bone marrow niche. The key findings and references are summarized in Table 2. 9.1. Vitamin D supplementation and arterial stiffness Vitamin D is a fat-soluble, steroid hormone that can be synthesized in the skin through

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ultraviolet exposure and is an essential hormone for calcium homeostasis (Lutsey & Michos, 2013). Vitamin D deficiency is associated with osteoporosis and bone fracture, especially in the elderly. Besides its role in bone health, several lines of evidence suggest that vitamin D could also play a critical role in maintaining cardiovascular function. While vitamin D deficiency is pandemic with more than 50% of the world pop ulation having inadequately lower level, observational studies suggest that decreased vitamin D levels are associated with an increased burden of arteriosclerosis (Kassi et al., 2013). Observational studies also showed that a low level of serum vitamin D is associated with increased cardiovascular mortality

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(Giovannucci et al., 2008; Zhao et al., 2012). Vitamin D deficiency is also linked to

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progressive organ damage such as heart failure and dementia (Anderson et al., 2010; Littlejohns et al., 2014; Sommer et al., 2017). Vitamin D is known to show favorable effect

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on endothelial dysfunction, VSMC migration/proliferation and inflammatory cell modulation through vitamin D receptors on various cell types (Kassi et al., 2013). Vitamin D can

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significantly induce nitric oxide production in endothelial cells by activating endothelial

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nitric oxide synthase (Molinari et al., 2011). Vitamin D not only retards VSMC growth, but also inhibits the expression of plasminogen activator inhibitor-1 in VSMCs (Mitsuhashi et al., 1991; Wu-Wong et al., 2006). Moreover, it exerts a beneficial effect on glucose metabolism,

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lipid profile, and renin-angiotensin-aldosterone system, thereby protecting vessel walls and

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reducing arterial stiffness (Kassi et al., 2013). Investigation of stroke patients demonstrated that vitamin D deficiency is prevalent among Asian stroke patients, and decreased serum

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vitamin D levels are independently correlated with various markers of cerebral small vessel disease, such as cerebral microbleeds, old lacunes, and white matter hyperintensities (Chung et al., 2015). Serum vitamin D is also an independent predictor of functional outcome after stroke (Park et al., 2015), and its dietary intake seems to be inversely related with mortality among Asian stroke patients (Sheerah et al., 2018). However, randomized clinical trials failed to demonstrate protective effects of vitamin D supplementation for the general healthy population to prevent vascular events (Shapses & Manson, 2011; Arora & Wang, 2017; Zittermann, 2018). A recent Mendelian randomization study showed that vitamin D level is inversely related with elevated blood pressure (Vimaleswaran et al., 2014). Moreover, small group studies focusing on subjects with a vitamin D deficiency showed that vitamin D supplementation could reduce arterial stiffness as measured by carotid femoral pulse wave velocities (Al Mheid et al., 2011; Raed et al., 2017). In this regard, a targeted strategy of vitamin D replacement for those patients with increased arterial stiffness and deficient serum

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vitamin D levels may be necessary to examine its vascular protective potential. 9.2. Bisphosphonate/denosumab and vascular calcification Bisphosphonates are analogues of inorganic PP and inhibit bone resorption by inducing osteoclast apoptosis, thereby preventing deterioration of bone architecture (Poole & Compston, 2012). The effectiveness of bisphosphonate treatment in fracture prevention and overall safety has been demonstrated by several randomized clinical trials in both sexes, and some studies showed an overall survival benefit beyond the reduced chances of fracture,

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suggesting a possible vascular protective effect (Poole & Compston, 2012; Lyles et al., 2007;

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Reid et al., 2018). For example, randomized clinical trials investigating the fracture prevention effect of zoledronate among older women with osteopenia and hip fracture

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patients demonstrated fewer cardiovascular events among zoledronate-treated group than control group (Lyles et al., 2007; Reid et al., 2018). A recent meta-analysis including

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randomized clinical trials revealed that bisphosphonates reduce arterial wall calcification, but

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not arterial stiffness or cardiovascular events (Kranenburg et al., 2016). The vascular protective effect of bisphosphonate could be multifactorial. Since PP is a strong inhibitor of vascular calcification and bisphosphonate is a PP analogue, it is reasonable to assume that

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bisphosphonate inhibits vascular calcification (Elmariah et al., 2010; Kranenburg et al., 2016).

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Bisphosphonate can modulate cholesterol biosynthesis by blocking farnesyl diphosphate synthase, thereby reducing overall cholesterol levels (Guney et al., 2008). Animal studies

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have shown that bisphosphonate treatment could reduce the progression of arterial vascular calcification by blocking apatite hydroxyapatite crystal formation in vessel walls (Lomashvili et al., 2009; Caffarelli et al., 2017). Another vascular protective mechanism can be due to restoration of the bone marrow niche by bisphosphonate. An animal study reported that zoledronate treatment significantly increases bone volume and HSC mass, as well as upregulates genes supporting the osteoblastic niche (Soki et al., 2013). However, intravenous zoledronate treatment in clinical trials was associated with increased incidence of new-onset atrial fibrillation, which may result in cardioembolic stroke (Black et al., 2007; Herrera et al., 2015). Denosumab is a human monoclonal antibody against RANKL for the treatment of osteoporosis and shows more potent effects in preserving bone mineral density compared with bisphosphonate in randomized clinical trials (Cummings et al., 2009). Denosumab

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showed a protective effect against atherosclerosis progression and an inhibitory effect against vascular calcification in animal studies (Helas et al., 2009). However, the analysis of 2,363 postmenopausal women with osteoporosis selected from 7808 participants in the Fracture Reduction Evaluation of Denosumab in Osteoporosis Every 6 Months trial study showed that denosumab treatment had a neutral effect on the progression of aortic calcification or vascular events (Samelson et al., 2014). 9.3. Romosozumab and cardiovascular safety

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Romosozumab is a monoclonal antibody that binds and inhibits sclerostin, which is a

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negative regulator of bone formation and is known to inhibit Wnt signaling and downregulate osteoblast development (Cosman et al., 2016). Recent randomized clinical trials

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demonstrated that romosozumab treatment was associated with a lower risk of clinical fracture and sustained bone mineral density better than placebo (Cosman et al., 2016; Saag et

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al., 2017). However, romosozumab treatment resulted in more cardiovascular serious adverse

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events (2.5%) than alendronate treatment (1.9%) at 12 months (Saag et al., 2017). The increased number of cardiovascular events after romosozumab treatment might be due to either its detrimental effect on the cardiovascular system or the protective effect of

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alendronate. Patients with chronic kidney disease who have vascular and aortic calcifications

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have high serum sclerostin levels, and sclerostin is expressed in vascular tissue undergoing calcification, possibly to suppress the progression of vascular calcification (Lim et al., 2017).

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Although sclerostin shows calcification inhibitory effects in vivo studies, a clinical study demonstrated that high sclerostin levels are related to cardiovascular mortality (NovoRodríguez et al., 2018).

9.4. PTH analogues and the bone marrow niche Teriparatide is a recombinant protein consisting of human PTH [1-34], and it was reported to restore bone architecture and reduce fracture risk in patients with osteoporosis (Shao et al., 2003). Several animal studies have shown that teriparatide treatment retards vascular and valvular calcification (Shao et al., 2003; Hsu et al., 2018). Teriparatide inhibited osteogenic vascular calcification in the aortic vessel walls of diabetic LDL receptor-deficient mice (Shao et al., 2003). In aged apolipoprotein E-deficient mice, teriparatide treatment resulted in reduced

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F-NaF incorporation at sites of aortic calcification (Hsu et al., 2018). A clinical

study demonstrated that teriparatide treatment increased the production and mobilization of

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peripheral HSCs in postmenopausal women when treatment persisted for 3 months, possibly by restoring the bone marrow microenvironment (Yu et al., 2014). Abaloparatide is a PTH-related protein analogue drug with 41% homology to PTH, and exerts bone anabolic potential by selectively binding to the RG conformation of the PTH type 1 receptor (Tella et al., 2017; Miller et al., 2016). It showed a somewhat greater bone anabolic effect than teriparatide from clinical studies (Miller et al., 2016), but its effects on the cardiovascular system and arteriosclerotic calcification have not yet been reported.

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9.5. Selective estrogen receptor modulator Estrogen plays a fundamental role in skeletal growth and bone homeostasis in both men and

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women (Weitzmann & Pacifici, 2006). Estrogen replacement therapy also exerts a cardioprotective effect by lowering cholesterol and blood pressure, but it is associated with an

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increased risk of thromboembolic events. The term ‘selective estrogen receptor modulator’ designates compounds that have tissue-specific estrogen agonist/antagonist properties, such

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as raloxifene. These compounds show estrogen agonist effects in some tissues and estrogen antagonist effects in others because of different ligand structures, receptor binding affinities,

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intracellular pathways and receptor subtypes (Saitta et al., 2001a). Raloxifene has estrogen agonist effects on bone and lipids and estrogen antagonist effects on the breast and uterus,

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thereby increasing bone mineral density and lowering cholesterol levels at the same time

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(Saitta et al., 2001a). Raloxifene therapy also improves flow- mediated endothelium dependent vasodilation (Saitta et al., 2001b). However, a randomized clinical trial with raloxifene targeting breast cancer risk as well as cardiovascular events did not show a cardioprotective effect (Barrett-Connor et al., 2006). Although treatment with raloxifene for a median of 5.6 years reduced the incidence of breast cancer, it did not reduce coronary events and significantly increased the risk of fatal stroke and venous thromboembolism (BarrettConnor et al., 2006). 9.6. Anti-inflammatory therapeutics Although inflammation is a vital step for atherosclerosis progression and several p reclinical studies confirmed the promising effects of inflammation modulating treatments, few therapeutic options based on anti- inflammation strategy have proved efficacy in clinical use. However, a recent study demonstrated that a selective anti- inflammatory strategy against

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innate immunity using a monoclonal antibody blocking the interleukin (IL)-6 pathway could effectively decrease the recurrence of atherosclerotic vascular events without modulating the lipid profile (Ridker et al., 2017). Canakinumab, a therapeutic monoclonal antibody against IL-1β, effectively reduced the levels of high-sensitivity C-reactive protein and IL-6, and treatment was associated with higher fatal infection and lower cancer incidences (Ridker et al., 2017). The same study group examined another anti- inflammatory strategy with low dose methotrexate in parallel, which has a more affordable price compared with canakinumab, but did not show clinical benefits in cardiovascular event prevention and had several notable

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complications (Ridker et al., 2019). While the inflammation modulating mechanism is still

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unclear, low-dose methotrexate did not reduce the levels of inflammatory cytokines, but increased the incidence of cancer, infection and leukopenia (Ridker et al., 2019). Besides the

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difference of inclusion criteria according to inflammatory biomarker profile and specificity of the therapeutic agents between the two trials, the detrimental effects on the bone marrow

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niche might also play a role in the differential effect on cardiovascular events. Methotrexate

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is known as major pharmacologic agent with bone marrow suppression potential, even at low doses (Sosin & Handa, 2000). A defective bone marrow niche induced by methotrexate treatment may also have exerted a progressive negative impact on vascular integrity and

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restorative capacity. On the other hand, an animal study with glucocorticoid- induced

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osteoporotic mice showed that IL-6 contributed to the defective osteogenesis of bone marrow stromal cells and neutralizing IL-6 by antibody administration restored the osteoporotic

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phenotype (Li et al., 2016). Elevated serum IL-6 was reported to be a major predictor of bone loss among postmenopausal women (Scheidt-Nave et al., 2001), suggesting canakinumab might have favorable effect on the bone marrow niche. 9.7. Summary of therapeutic strategies Currently, there are diverse therapeutic options for bone mineral loss based on different mechanism, and their effects on atherosclerosis and overall vascular integrity are heterogeneous. Bisphosphonates are one of the most commonly prescribed medications for osteoporosis and may harbor anti-atherosclerosis activity. Since no clinical studies have been conducted to study vascular effects as a primary outcome of bisphosphonate treatment, therapeutic application of bisphosphonate cannot yet be extended to the prevention of atherosclerotic events in general (Caffarelli et al., 2017). However, it seems plausible to consider its use for older patients with both atherosclerotic disease and high fracture risk.

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Some therapeutic agents such as estrogen or selective estrogen reuptake modulators are known to increase thromboembolic events, requiring careful considerations when prescribing for patients with increased stroke risk. Meanwhile, more observational studies focusing on cardiovascular events after treatment with PTH analogues or anti-sclerostin antibody therapy, and Mendelian randomization studies are required to outline the mechanistic link between arteriosclerosis and bone mineral loss. 10. Conclusion Current treatment strategies against atherosclerosis generally focus on lowering the LDL

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cholesterol levels and modifying other cardiovascular risk factors, which has proven to be effective in suppressing atheroma growth and reducing atherosclerotic vascular events, and

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this strategy is based on ‘the lower, the better’ principle. However, a considerable number of patients still suffer from atherosclerotic events despite being on the highest possible dose of

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the most effective treatment. Moreover, ‘zero LDL cholesterol’ for the prevention of atherosclerosis progression might not be an achievable target for living organisms. Increased

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atherosclerosis and vascular calcification along with decreased bone mineral density is prevalent among the older population, which might be an inevitable consequence of the

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increased entropy principal for a closed vital system. Novel treatment options based on the

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bone-vascular axis might be a promising avenue for the prevention of vessel wall degeneration, subsequent target organ damage and overall survival by preventing fracture.

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Harmonizing the bone marrow microenvironment to sustain the stem cell compartment might be helpful to maintain vascular health and regeneration capacity. Minimizing inflammatory cell recruitment and detrimental phenotype switching within the atheroma is critical among patients with recent atherosclerotic events. Maximizing cellular signals related to the healing process for vessel walls and target organ damage is essential for patients with previous atherosclerotic vascular events or with advanced vessel wall degeneration.

Conflict of interest statement: The authors declare that there are no conflicts of interest. Acknowledgment:

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This study was supported by grants of the Basic Science Research Program through the National Research Foundation of Korea (2017R1A2B4005854, 2018R1D1A1B07048484, and 2019R1F1A1059455). The funding source has no role in design, collection, analysis or interpretation of data; in the writing of the manuscript; and in the decision to submit the

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manuscript for publication.

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Figure legends Fig. 1. Interaction between bone and vascular architecture (A) Normal bone-vascular axis with intact inhibitory signals against calcification and an adequate supply of EPCs is necessary to maintain vessel wall integrity. (B) Pathologic bonevascular axis leading to atheromatous plaque progression with intima spotty calcification is accompanied by increased production of inflammatory cells from the bone marrow. (C) Pathologic bone- vascular axis leading to severe arteriosclerotic calcification shows

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exaggerated calcification signals within vessel wall and increased number of circulating

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calcifying cells recruited from bone marrow.

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Fig. 2. The application of positron emission tomography for patients with cerebral atherosclerosis

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(A) Subjects with cerebral atherosclerosis exhibited lower FDG uptake at lumbar vertebrae

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than those without cerebral atherosclerosis (black arrows), although the FDG uptake level in the spleen was not significantly different between the two groups. Reproduced with from

fluorodeoxyglucose

“Decreased

bone

positron

emission

marrow

al

permission

activity

tomography

measured

by

among patients

using with

18F-

cerebral

rn

atherosclerosis” by Kim, J. M. et al, 2019, Journal of Neurosonology and Neuroimaging, in

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press, 2019 by Korean Society of Neurosonology. (B) When radiolabeled FDG and NaF ligands were applied to detect unstable atheroma among stroke patients with carotid atherosclerosis, both FDG and NaF radiotracer signals were prevalent, reflecting high inflammation and calcification burden. A patient with left middle cerebral artery infarction and culprit left carotid atheroma (red arrows) showed strong FDG uptake and minimal NaF uptake (upper panel). A patient with right middle cerebral artery infarction had non-culprit left carotid atheroma (yellow arrows) which showed minimal FDG uptake and increased NaF uptake (lower panel). Reproduced with permission from “Analysis of 18F- fluorodeoxyglucose and 18F- fluoride positron emission tomography on stroke patients with carotid atherosclerosis” by Kim, J. M. et al, 2019, Journal of Lipid and Atherosclerosis, in press, 2019 by the Korean Society of Lipid & Atherosclerosis.

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Table 1. Connecting mediators between bone and vessel wall Mediators

Bone

Blood vessel

Osteoprotegerin

Glycoprotein,

Inhibits osteoclasts by -

Inhibits

(OPG)

member of TNF acting as a decoy calcification (Bucay et al., receptor

receptor for RANKL 1998)

superfamily

(Boyle et al., 2003)

-

member 11B

May

medial

induce

vascular

calcification

and

atherosclerosis (Toffoli et

oo

f

al., 2011) -

Positive

correlation

e-

pr

between serum OPG levels and

vascular

calcification/cardiovascular mortality (Tschiderer et al.,

Pr

2018)

Pyrophosphate

Potent inhibitor of Inhibits mineralization - Inhibits calcium crystal

(PP)

calcium phosphonate

Bellosta

Matrix

G1a

protein (MGP)

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rn

precipitation

al

of osteoblasts (Villa- formation (O’Neill et al.,

Mineral-binding proteins binds

&

Egido,

2017)

2011) -

Negative

correlation

between PP

levels and

arterial

stiffness

(Lomashvili et al., 2005) Inhibits

abnormal -

Inhibits

medial

which mineralization (Luo et calcification (Luo et al., to

BMP2 al., 1997)

1997)

when activated by

-

MGP

vitamin K

associated with risk factors for

increase

is

atherosclerosis

(Hermans et al., 2007) Fetuin-A

Liver-derived

An inhibitor of the

-

glycoprotein

formation

calcification

and

precipitation of apatite

Inhibits

medial (Jahnen-

Dechent et al., 2011)

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precursor

mineral -

(Jahnen-Dechent

Negative

et between

al., 2011)

correlation

fetuin-A

vascular

and

calcification,

atherosclerosis, inflammation, malnutrition and mortality (Wang et al., 2005) Non-collagenous

(OP)

bone

Inhibits osteoclasts by - Localized in calcified

matrix binding (Anagnostis

et

proteins

and

osteogenesis

(BMPs)

(Anagnostis

et

al.,

al rn Jo u growth signaling

associated with increased burden

(Dhore et al., 2001; Zhang

- Upregulated in atheroma specimens (Chen et al., 2006) Renal -

Klotho/FGF23/FGF

- Inhibits insulin receptor and

Wolak, 2014)

et al., 2015)

- Cofactor of FGF receptor

calcification

atherosclerosis

2009)

Klotho

vascular

osteoblast - Elevated BMP levels are

differentiation

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morphogenetic

Promotes

e-

TGF-β superfamily

al., 2009). - May attenuate or worsen

pr

2009)

Bone

al.,

oo

glycoprotein

integrins atheroma (Anagnostis et

f

Osteopontin

insulin- like are

exhibit

complexes

essential

Klotho-deficient

mice

impaired

angiogenesis and

medial

for calcification (Kuro-O et al.,

factor-1 vitamin D metabolism 1997) and

phosphate

- Low circulating Klotho

homeostasis

levels are associated with

- Reduces osteogenic

macrovascular

capacity

among type

and

osteoclastogenesis (Kurosu et al., 2005)

outcomes 2

diabetic

patients (Pan et al., 2018)

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-

Upregulates OPG

expression miR-17

- Targets Smad7 in MSCs

Suppresses - Positively associated with

osteoblastic

-

unstable

atheroma

Targets differentiation and cell coronary and

connective

tissue proliferation (Jia et al.,

growth factor and 2014)

in

cerebral

arteries (Chen et al., 2015; Kim et al., 2015)

thrombospondin-1 - Promotes osteoblast Upregulated cell differentiation

death 4 protein

mineralization, inhibits

and

cardiac

hypertrophy and fibrosis in

oo

programmed

in

f

Targets

but response

to

pressure

overload (Small & Olson,

pr

miR-21

osteoclastogenesis

2011)

RUNX2 - Inhibits osteogenic

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Targets and Osterix

differentiation

rn

al

2017)

Inhibits

VSMC

of calcification (Goettsch et

MSCs (Wang et al.,

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miR-125b

e-

(Gennari et al., 2017)

al., 2011)

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Therapeutic Vitamin

Bone

D

soluble

Blood vessels

(fat - Maintains bone mineral -

steroid

hormone

density

by

Favorable

bone uptake

architecture)

from

intestines

on

endothelial

increasing dysfunction, vascular smooth muscle cell

that calcium and phosphate proliferation,

regulates

effects

and

inflammatory

cell

the modulation (Kassi et al., 2013)

(Lutsey

& - Neutral effect on vascular health in large-

Michos, 2013)

sized randomized clinical trials (Shapses &

oo

f

Manson, 2011)

- Reduced arterial stiffness among vitamin

pr

D deficient patients with coronary artery disease (Al Mheid et al., 2011)

- Inhibits bone resorption - May reduce vascular calcification and

(stable

by inducing osteoclast arterial stiffness (Elmariah et al., 2010;

a

naturally

apoptosis

(Poole

& Kranenburg et al., 2016)

Pr

of

analogues

e-

Bisphosphonates

Compston, 2012)

pyrophosphate)

- Prevents deterioration pathway, reducing overall cholesterol levels of

bone

Modulates

cholesterol

biosynthesis

&

architecture (Guney et al., 2008) Compston, - Restores bone marrow niche and HSC

rn

(Poole

-

al

occurring

mass (Soki et al., 2013)

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2012)

- Some bisphosphonates are associated with increased risk for development of atrial fibrillation (Black et al., 2007; Herrera et al., 2015)

Denosumab

(a - More potent effect at -

Protective

against

atherosclerosis

human monoclonal preserving bone mineral progression and vascular calcification from antibody

that density

inhibits RANKL)

than animal models (Helas et al., 2009)

bisphosphonates

-

(Cummings et al., 2009)

calcification or vascular events in post-hoc

-

Binds

decreases

RANK

No

significant

effect

on

aortic

and analysis of human trials (Samelson et al.,

osteoclast 2014)

formation and activity

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(Cummings et al., 2009) Romosozumab

(a - Potent effects on bone - Sclerostin levels are increased in chronic

monoclonal

formation

antibody

and kidney

that preservation

inhibits sclerostin)

mineral

of

patients,

possibly

to

bone suppress vascular calcification (Lim et al.,

density

inhibiting

disease

by 2017)

sclerostin, - More cardiovascular adverse events

which suppresses Wnt reported

after romosozumab

treatment

signaling (Cosman et al., compared with bisphosphonates (Saag et al., 2017)

f

2016)

(a - Increases bone mineral - Inhibits aortic calcification and vascular

recombinant

density

anabolic calcifying activity in LDL receptor (-/-) and

[1- effects (Shao et al., 2003) Apo E (-/-) mice (Shao et al., 2003; Hsu et

pr

human PTH

with

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Teriparatide

34])

al., 2018)

e-

- Possibly restores bone marrow niche,

Pr

increasing production and mobilization of HSCs (Yu et al., 2014)

Selective estrogen - Inhibits bone resorption (Saitta et al., 2001a)

with

tissue-selective estrogen

-

receptor

agonist

LDL

Increases

fatal stroke and

venous

thromboembolism (Barrett-Connor et al.,

Jo u

(compounds

cholesterol,

2001a)

rn

modulators

total

cholesterol and homocysteine (Saitta et al.,

al

receptor

Decreases

2006)

or

antagonist activity) Canakinumab

- May reduce the risk of Reduces cardiovascular events, particularly

(fully

fracture

humanized

in for those with the highest reduction of C-

anti- interleukin-1β

glucocorticoid- induced

reactive protein (Ridker et al., 2017)

monoclonal

osteoporosis

antibody)

inhibiting interleukin 6

by

signaling pathway Table 2. Therapeutic modalities that may have promising effects on vascular disease based on the bone-vascular axis

Figure 1

Figure 2