Sergio Bertazzo

Shweta Agarwal

Comptes Rendus Chimie

lable at ScienceDirectC. R. Chimie 19 (2016) 1605e1609Contents lists avaiComptes Rendus Chimiewww.sciencedirect.comAccount/RevueNew paradigms in cardiovascular calcificationShweta Agarwal a, Sergio Bertazzo b, *a Department of Materials, South Kensington Campus, Imperial College, London, SW7 2AZ, UKb Department of Medical Physics & Biomedical Engineering, University College London, Malet Place Engineering Building, London, WC1E6BT, UKa r t i c l e i n f oArticle history:Received 27 February 2015Accepted 29 September 2015Available online 21 February 2016Keywords:Vascular calcificationBiomineralisationElectron microscopyCalciumPhosphate* Corresponding author.E-mail addresses: s.agarwal12@imperial.ac.uk (S.ucl.ac.uk (S. Bertazzo).http://dx.doi.org/10.1016/j.crci.2015.09.0131631-0748/© 2015 Académie des sciences. Publishea b s t r a c tOut of several concurrent hypotheses that attempt to explain the development of calcificlesions in vascular tissues, the most widely accepted is based on the formation of bone-liketissue. Conversely, a study recently published on a materials science journal shed new lighton the nature of cardiovascular calcification. The results have shown that cardiovascularcalcification found in tissues affected by different calcific diseases is formed from the samenano/micro calcified spherical particles, which are also the first calcified structure thatcould be detected in vascular tissue. Moreover, cardiovascular calcification is not formedmainly by bone tissue but, surprisingly, by a not previously described, highly crystallinecalcium phosphate material. By combining biology, materials science and chemistry, thenew results have generated fundamental insights into the nature of cardiovascular calci-fication. This perspective summarizes these findings and epitomizes the scientificapproach it has used to address important clinical questions.© 2015 Académie des sciences. Published by Elsevier Masson SAS. All rights reserved.1. IntroductionCalcification in the cardiovascular system is a commonmineral metabolic disease that contributes to many of theapproximately 17 million deaths per year caused by car-diovascular disease [1]. The burden of calcific disease isrelatively larger in male individuals and increases withadvancing age, diabetes mellitus, hypertension, smoking,elevated low-density lipoprotein levels, metabolic syn-drome and renal dysfunction [2]. The cardiovascular tissuesmore commonly affected include the aorta, femoral arteriesand aortic valves [3e5]. In cardiac valves, calcification ischaracterized by calcific lesions on the valve cusp, resultingin an impaired movement of the leaflets. Pathologicalmineralization in arteries can lead to stenosis throughAgarwal), s.bertazzo@d by Elsevier Masson SAS. Athickening and stiffening of tissues, reducing blood flowand eventually resulting in heart failure [6,7].Calcification associated with atherosclerosis maysignificantly increase health concerns. Calcification nucleihave been associated to cell death and atherosclerotic intraplaque hemorrhages. The differences in mechanical prop-erties between the mineral and the tissue can actuallyprovoke hemorrhages. Moreover, the calcification found inthe lipid core of lesions can produce micro fractures, whichin turn can lead to acute thrombosis and even fatalmyocardial infarction [8,9].2. Existing models of cardiovascular calcificationCardiovascular calcific disease was described as a pas-sive degenerative process that occurs due to deteriorationof tissues caused by the stress and strain of cardiac func-tion. More recently, research associated with molecularsignaling processes of vascular homeostasis has changedthis view of cardiovascular calcification and redefined it asll rights reserved.Fig. 1. The topographical and compositional information gathered from secondary electron and backscattering signals with SEM are correlated by color-codingand overlapping, generating density-dependent color images (DDC) that highlighted the location of nano-scale calcified features within the organic matrix. Theorange color identifies denser material; structures that appear green are less dense. DDC-SEM micrographs of the aortic valve presenting (a and b) densespherical particles (scale bars ¼ 1 mm), (c) dense fibers (scale bar ¼ 1 mm), and (d) compact material (scale bar ¼ 10 mm).S. Agarwal, S. Bertazzo / C. R. Chimie 19 (2016) 1605e16091606an active process [5]. This active remodeling process isthought to involve inflammation and calcification due totransdifferentiation of cardiovascular cells (such as vascularsmooth muscle cells and valve interstitial cells) intoosteoblast-like cells [10e12]. Although cellular disease-driven processes can be targeted therapeutically, thereare currently no drug therapies developed specificallyagainst cardiovascular calcification [13]. The only treatmenttoday is surgery and, if the aortic or mitral valve is affected,this means the surgical replacement of the valve. Regret-tably, the recurrent calcification of bioprosthetic valves stillcarries an operative mortality of around 50% within 10years following surgery [4,5,14].2.1. New perspectiveRecently, materials characterization techniques havebeen applied to cardiovascular calcification [15e17] insamples of aortic valve tissue, mitral valves, coronary ar-teries and aortas from rheumatic fever, aortic valve stenosisand atherosclerosis patients. Scanning electron microscopy(SEM) revealed the micro morphology of these samples,showing that calcific lesions (the macroscopic calcific de-posits found in diseased tissue) can include three distinctcalcified structures: spherical particles, calcified fibers andcompact calcification (Fig. 1aed).Energy-dispersive X-ray spectroscopy (EDS) furtherrevealed that all three structures are composed of calciumand phosphorus, with the spherical particles also contain-ing magnesium (Fig. 2). The elemental composition istherefore an indication that the different structures mighthave different origins.With regard to tissue morphology, even if the calcifiedfibers in calcific lesions displayed some resemblance to thecalcified fibers in bone, spherical particles with the size re-ported (between 100 nm and 10 microns) have never beendescribed in bone tissue studies. The spherical particlesreported recently were found on macroscopically visiblecalcific lesions, in regions of aortic valves with no visiblecalcific lesions but where calcificationwas found elsewhereis the vascular system, and in aortic valves with no visiblecalcific lesions and where the vascular system examinedwas free of any calcific lesion. Unlike the calcified spheres(Fig. 1a and b), dense fibers (Fig. 1c) and compact calcifiedmaterials (Fig. 1d) were only identified in association withcalcific lesions and never in non-calcified tissues.When the calcific lesions were sectioned by Focused ionbeam (FIB) and imaged by Transmission electron micro-scopy (TEM) [18], spherical particles were found not onlyon the surface of calcific lesions, but also embedded withinlesions. This is an indication that this calcification process isnot a clear-cut surface precipitation of calcium phosphate,but rather a more complex biomineralization process thatoccurs in the bulk of the tissue.Another important piece of information provided byelectron microscopy analysis is that spherical particles arealso present inside the compact calcification. This meansthat the spherical particles keep their original morphologyand internal structure while embedded in another calcifiedstructure, suggesting in this way that calcific lesions mightbe formed by more than one process of biomineralization,and that the growth of compact calcification structurespossibly engulfs spherical particles, which are the mostconspicuous and earliest noticeable calcified structures invascular tissue.Interestingly, the internal structure of calcified sphereswas organized in rings, similarly to the internal structure ofa tree trunk. Crystallographic analyses also provided one ofFig. 2. DDC-SEM micrographs of the aortic valve presenting dense spherical particles (a), dense fibers (b) and the compact material (c). The corresponding EDSspectra collected at the marked () sites are indicated on micrographs aec, respectively. Scale bars ¼ 2 mm.S. Agarwal, S. Bertazzo / C. R. Chimie 19 (2016) 1605e1609 1607the most extraordinary results, by demonstrating that thespherical particles were formed from highly crystallinehydroxyapatite.The fact that these spheres can have up to 5microns anddiffract as a single crystal, makes them (to the best of ourknowledge) the most crystalline mineral present in verte-brates. On the other hand, the compact calcification pre-sented a poorly crystalline apatite, which is surprisinglyeven less crystalline than bone mineral (Fig. 3). Once again,the results indicate there are relevant chemical differencesbetween the structures of the material from calcific lesionsand bone.Fig. 3. TEM image showing spherical particles trapped in compact organic calciumdifferent regions of the tissue section are numbered 1e2 (scale bars ¼ 0.05 nm). Smaterial (1), whereas the surrounding compact material shows diffraction patternsFinally, perhaps one of the most relevant findings is thatthe morphological and chemical characteristics of thecalcific material resulting in rheumatic fever, atheroscle-rosis and aortic valve stenosis are roughly the same. Thisconclusion is supported, firstly, by the finding that thecalcified material in all three diseases is formed fromcalcified spherical particles, calcified fibers and compactcalcification. Moreover, samples from all different diseasespresented roughly the same morphology, the sameelemental composition and, even more strikingly, the samecrystallinity. This is particularly true for the spherical par-ticles, which present the same internal ring-shapedphosphate matrices (scale bar ¼ 0.5 mm). The SAED patterns obtained frompherical particles produce diffraction patterns typical of a highly crystallinetypical of a poorly crystalline material (2).S. Agarwal, S. Bertazzo / C. R. Chimie 19 (2016) 1605e16091608structure, the same amount of magnesium in theircomposition and the same electron diffraction pattern of asingle crystal across samples of tissue affected by differentcalcific diseases.Taken together, these findings provide strong evidencethat three distinct calcific diseases (rheumatic fever,atherosclerosis and aortic valve stenosis) present a relevantcommonality in their process of calcification. In this way,since all of them are formed by the same biomineral, theymight even present the same mechanism of biominerali-zation. At this stage it is not possible to know if the origin ofthe three diseases is the same, but it is clear that oncestarted, then biomineralization process produces the samematerial.Even if these results are not able to provide insights intothe biochemical or cellular mechanism by which the min-eral is formed, they give important clues as to wherefurther research is more likely to discover the origin ofpathological calcification in the vascular system. The in-formation provided urges the field to look for systemswhere it is possible to produce calcium phosphate mineralswith specific morphology, composition and crystallinity.The literature contains suggestions for the origin ofcalcified material found in the tissue, such as stromal cellmicrovesicles and apoptotic bodies[19]. Confirming thesehypotheses or even finding unexpected origins of thesecalcified structures found in the cardiovascular system, is anecessary step to a better understanding of calcific disease.This knowledgewill at the same time potentially unlock thedevelopment of new treatment and prevention methods.3. Comparison to osteogenic theory of cardiovascularcalcificationConsidering that the typical bone mineral is presentalong collagen fibers, formed by poorly crystalline apatiteand composed of plate-like structures with a typical size of20 nm [20,21] whilst calcified particles from calcific lesionsin vascular tissue are highly crystalline and with sizesstarting at 100 nm, it is clear that the material found invascular calcification is not bone.Bone formation could nevertheless occur in vasculartissue either as a concomitant event or as a consequence ofthe formation of calcified particles, since themere presenceof hard spherical particles could trigger mesenchymal stemcell differentiation to osteoblast phenotype [22,23]. It isnow well documented that in contact with practically anytissue, calcium phosphate can lead local cells to a process oftransdifferentiation into mineralized phenotype [24e27].Although the mechanism by which the presence ofcalcium phosphate initiates the formation of a bone-likematerial remains unclear, some studies provide evidencethat valve interstitial cells may contribute to or even beresponsible for the formation of calcific lesions [28].This hypothesis might also explain why there are somany results in the literature demonstrating the presenceof bone cells and bone proteins in calcific lesions. In thisway, the presence of these cells and proteins would indeedindicate a correlation with bone formation, but does notoffer proof that the process of calcification is triggered bythe presence of bone cells, which could have arrived afterinitial stages of calcification, when the presence of calciumphosphate leads to the recruitment of bone cells or differ-entiation of vascular cells to bone cells in the calcific lesion.4. ConclusionTo date, cardiovascular calcification has been regardedas an actively regulated process, even though the mecha-nisms associated with late stage mineralization remainlargely unknown. New evidence shows that vascularcalcification is not only a simple process of bone formation,but also includes another biomineralization process thatleads to the formation of a different material hitherto un-known. More important than just shedding light onto thematerial that forms cardiovascular calcification, these newresults urge the field to look for new ways to understandand describe vascular calcification as a process differentfrom osteogenic formation.Finally, the importance of an interdisciplinary approachto study biomedical questions such as vascular calcificationis clear. This approach provides a stepping-stone for clinicalinvestigation into calcific disease that will have a directimpact on the potential development of much-neededtreatments for vascular calcification.AcknowledgmentsShweta Agarwal was supported by the British HeartFoundation Research Excellence Award.References[1] D. Butler, Nature 477 (2011) 260e261.[2] A.H. Chester, Aswan Heart Centre Sci. Pract. Ser. 2011 (2011) 4.[3] F. Herisson, M.-F. Heymann, M. Chetiveaux, C. Charrier, S. Battaglia,P. Pilet, T. Rouillon, M. Krempf, P. Lemarchand, D. Heymann,Y. Gou€effic, Atherosclerosis 216 (2011) 348e354.[4] B. Iung, A. 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New paradigms in cardiovascular calcification

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New paradigms in cardiovascular calcification

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