Diabetologia (2014) 57:2251–2260 DOI 10.1007/s00125-014-3348-z REVIEW RANKL–OPG and RAGE modulation in vascular calcification and diabetes: novel targets for therapy Agbor Ndip & Fiona L. Wilkinson & Edward B. Jude & Andrew J. M. Boulton & M. Yvonne Alexander Received: 1 April 2014 /Accepted: 17 July 2014 /Published online: 12 August 2014 # Springer-Verlag Berlin Heidelberg 2014 Abstract Type 2 diabetes is associated with increased cardio- and inflammation in diabetes. Several therapeutic strategies vascular morbidity and mortality and early vascular ageing. could have advantageous effects on the vasculature in patients This takes the form of atherosclerosis, with progressive vas- with diabetes, including targeting the RANKL and receptor cular calcification being a major complication in the patho- for AGE (RAGE) signalling pathways, since there has been genesis of this disease. Current research and drug targets in little success—at least in macrovascular outcomes—with con- diabetes have hitherto focused on atherosclerosis, but vascular ventional glucose-lowering therapy. There is substantial and calcification is now recognised as an independent predictor of relevant clinical and basic science evidence to suggest that cardiovascular morbidity and mortality. An emerging regula- modulating RANKL–RANK–OPG signalling, RAGE signal- tory pathway for vascular calcification in diabetes involves the ling and the associated proinflammatory milieu alters the receptor activator for nuclear factor κB (RANK), RANK natural course of cardiovascular complications and outcomes ligand (RANKL) and osteoprotegerin (OPG). Important novel in people with diabetes. However, further research is critically biomarkers of calcification are related to levels of glycation needed to understand the precise mechanisms underpinning these pathways, in order to translate the anti-calcification Agbor Ndip and Fiona L. Wilkinson are joint first authors. strategies into patient benefit. A. Ndip : A. J. M. Boulton Department of Medicine and Diabetes, Manchester Royal Infirmary, Keywords AGE . Charcot neuroarthropathy . Diabetes . Manchester, UK OPG . RAGE . RANKL . Review . Vascular calcification A. Ndip Department of Diabetes and Endocrinology, Warrington and Halton Abbreviations NHS Trust, Warrington, UK CKD Chronic kidney disease A. Ndip : E. B. Jude : A. J. M. Boulton GACI Generalised arterial calcification of infancy Centre for Endocrinology and Diabetes, Faculty of Medical and OPG Osteoprotegerin Human Sciences, University of Manchester, Manchester, UK RAGE Receptor for AGE κ * RANK Receptor activator for nuclear factor B A. Ndip ( ) κ Manchester Diabetes Centre, 193, Hathersage Road, RANKL Receptor activator for nuclear factor Bligand Manchester M13 0JE, UK VSMC Vascular smooth muscle cell e-mail: [email protected] F. L. Wilkinson : M. Y. Alexander Healthcare Science Research Institute, Manchester Metropolitan Historical perspective of vascular calcification University, Manchester, UK E. B. Jude The earliest recognition of vascular calcification was made in Department of Diabetes, Diabetes Centre, Tameside General a 5,300 year-old naturally mummified iceman discovered in Hospital, Ashton-Under-Lyne, UK 1991 in the Tyrolean Alps on the Austrian–Italian border, A. J. M. Boulton : M. Y. Alexander where radiological evidence showed the presence of aortic Manchester Academic Health Science Centre, Manchester, UK and carotid calcification. Vascular calcification was also 2252 Diabetologia (2014) 57:2251–2260 detected in the mummified remains of King Tutankhamun, the ab Egyptian pharaoh who died more than 3,000 years ago [1]. The association between cardiovascular mortality and vas- cular calcification has also been documented in ancient humans. Allam and collaborators performed CT imaging on Plaque 20 Egyptian mummies and demonstrated identifiable aortic Plaque and/or peripheral vascular tissue calcification [2]. Of note, calcification was present in 87% of those who died aged ≥45 years but only 25% of those who were <45 years of age cd when they died. Although a direct causal link cannot be claimed based on these findings alone, there appears to be Plaque an association between ageing, calcification and mortality risk. This review critically appraises published work in light of our own findings and focuses on novel biomarkers associ- ated with vascular calcification in diabetes, while briefly highlighting potential novel therapeutic targets. Fig. 1 Intimal and medial vascular calcification in peripheral arteries. Sections (6 μm) of tibial arteries, taken from below knee amputations The pathogenesis of vascular calcification (a–c), and internal mammary artery (d), from patients undergoing coro- nary artery bypass surgery, were stained with alizarin red to highlight areas of calcification. The internal mammary artery was used as a nega- We [3–8] and others [9–16] have shown that vascular calcifi- tive control as very little or no calcification is observed in this vascular cation is a regulated process whereby smooth muscle cells, or bed. Intimal vascular calcification is associated with atherosclerotic possibly cells recruited from the tunica adventitia or the pe- plaque [closed arrow heads in (a)and(b)], and medial vascular calcifi- ripheral blood, differentiate into an osteogenic phenotype and cation is deposited as spicules, which can eventually build up to form a block of calcified material in the medial layer [long arrows in (a), (b)and produce bone-associated proteins. Not only are bone-forming (c)]. (c) also shows uraemic arteriopathy, where the medial layer appears proteins, such as osteocalcin, alkaline phosphatase and bone as one block of calcified material around the circumference of the vessel. morphogenetic protein-2 and -4, upregulated during vascular Scale bar, 1,000 μm calcification, but inhibitors of bone matrix deposition are also elevated (osteopontin and matrix Gla protein) in order to regulating medial calcification in diabetes, where it frequently negate the detrimental effects of bone in the vessel wall. All coexists with osteopenia [30–32], remains largely unclear, but of these osteogenic factors, found to be upregulated in calci- reports suggest an important role for RANKL–RANK–OPG fied vessels from diabetic patients, have been extensively signalling [6, 33]. Valvular calcification is also a considerable reviewed elsewhere [10, 17–19]. clinical problem. It is thought that abnormal haemodynamics Vascular calcification occurs in distinct locations in vascu- may be the major contributor in this case, since it has been lar beds under different disease contexts and is defined ac- shown that the deposition of a calcified matrix is site-specific cordingly [20, 21]. There are four histoanatomic variants of [34] and that the leaflet wall shear stress differs in both vascular calcification, namely intimal/atherosclerotic calcifi- pulsatility and magnitude between the tricuspid and bicuspid cation, medial calcification (sometimes referred to as aortic valve leaflets [10]. Finally, uraemic arteriolopathy Mönckeberg’s sclerosis), valvular calcification (e.g. calcific (calciphylaxis), is characterised by the deposition of calcium aortic stenosis and prosthetic valve calcification), and uraemic within the walls of small and medium sized vessels in the arteriolopathy (also called calciphylaxis) as reviewed in detail dermis and in the subcutaneous tissue and typically occurs in elsewhere [22, 23]. Each has a distinct pathogenesis and people with CKD or on renal dialysis [35]. occurs in different disease states. Intimal calcification refers to the calcific deposits found within atherosclerotic plaques (Fig. 1a, b) and may be triggered by elevated oxidative stress Vascular calcification and genetics or inflammation [21, 23, 24]. Medial calcification, generally associated with chronic kidney disease (CKD) and diabetes, Epidemiological studies have revealed a genetic component to can be present in association with intimal plaque formation vascular calcification [36, 37], with differences being reported (Fig. 1c), but may occur independently of atherosclerosis, in the prevalence and severity of coronary artery calcification being preferentially deposited along the elastic lamina and between African-Americans and whites [38]. The findings of extracellular matrix [25–27]. While medial calcification in the study were attributed to genes associated with the CKD is considered to be a consequence of altered calcium mobilisation of calcium and the immune/inflammatory re- and phosphate mineral metabolism [28, 29], the mechanism sponse. Mutations in genes encoding inhibitors of Diabetologia (2014) 57:2251–2260 2253 calcification have influenced the incidence of mineral deposi- In Europe and the USA it has been recommended that tion. For example, mutations in ENNP1, which encodes an coronary artery calcification scores should be used to screen enzyme (ectonucleotide pyrophosphatase–phosphodiesterase for and classify cardiovascular risk status in the general pop- 1) that converts ATP to adenosine and pyrophosphate [37], ulation [45, 48]. Also, conventional tools for predicting car- results in a loss of enzyme activity causing generalised arterial diovascular disease, such as the Framingham risk score, have calcification of infancy (GACI) [37]. Although GACI is usu- shown limitations in people with intermediate risks. In fact, ally lethal, there have been reports of successful treatment coronary artery calcification has been suggested, among other with bisphosphonates