Elemental analysis insights into atherosclerotic calcification

Background. Atherosclerosis is frequently accompanied by an extensive calcification, yet the mechanisms behind its initiation and progression remain obscure. In particular, there is unclear whether large mineral deposits grow concentrically or by merging of microcalcifications. Aim of the study was to investigate calcium phosphate maturation during atherosclerotic calcification employing an elemental analysis approach. Material and methods. We collected 20 calcified atherosclerotic plaques excised during carotid endarterectomy. After being fixed in formalin and postfixed in osmium tetroxide, plaques were dehydrated and stained in uranyl acetate with the subsequent embedding into epoxy resin, grinding, polishing, and lead citrate counterstaining. Upon the sputter coating with carbon, we visualised the plaque microanatomy by means of backscattered scanning electron microscopy. Elemental analysis was carried out using energy-dispersive X-ray spectroscopy in the backscattered mode at high vacuum and 20 kV voltage. The analysis was performed at 11 radial points within the calcium deposit and 4 control points at ascending distance from the deposit. Results. Calcium to phosphate ratio differed between the calcium deposits in distinct plaques and also within the same plaque. We found a statistically significant correlation between calcium to phosphate ratio in the center and periphery of the calcium deposit. Areas with distinct electron density had different calcium to phosphate ratio; however, there was no clear correlation between these parameters. Conclusion. Correlation of calcium to phosphate ratio in the center and periphery of the calcium deposit suggests that its maturation develops from the center to the periphery rather than by merging of neighboring calcium deposits.

atherosclerosis, calcification, mineralization, elemental analysis, calcium phosphate, hydroxyapatite

1. Shi X., Gao J., Lv Q., Cai H., Wang F., Ye R., Liu X. Calcification in atherosclerotic plaque vulnerability: Friend or foe? Front. Physiol. 2020; 11: 56. doi: 10.3389/fphys.2020.00056

2. Kelly-Arnold A., Maldonado N., Laudier D., Aikawa E., Cardoso L., Weinbaum S. Revised microcalcification hypothesis for fibrous cap rupture in human coronary arteries. Proc. Natl. Acad. Sci. USA. 2013; 110 (26): 10741–10746. doi: 10.1073/pnas.1308814110

3. Petsophonsakul P., Furmanik M., Forsythe R., Dweck M., Schurink G.W., Natour E., Reutelingsperger C., Jacobs M., Mees B., Schurgers L. Role of vascular smooth muscle cell phenotypic switching and calcification in aortic aneurysm formation. Arterioscler. Thromb. Vasc. Biol. 2019; 39 (7): 1351–1368. doi: 10.1161/ATVBAHA.119.312787

4. Vengrenyuk Y., Carlier S., Xanthos S., Cardoso L., Ganatos P., Virmani R., Einav S., Gilchrist L., Weinbaum S. A hypothesis for vulnerable plaque rupture due to stress-induced debonding around cellular microcalcifications in thin fibrous caps. Proc. Natl. Acad. Sci. USA. 2006; 103 (40): 14678–14683. doi: 10.1073/pnas.0606310103

5. Zhan Y., Zhang Y., Hou J., Lin G., Yu B. Relation between superficial calcifications and plaque rupture: An optical coherence tomography study. Can. J. Cardiol. 2017; 33 (8): 991–997. doi: 10.1016/j.cjca.2017.05.003

6. Akers E.J., Nicholls S.J., di Bartolo B.A. Plaque calcification: Do lipoproteins have a role? Arterioscler. Thromb. Vasc. Biol. 2019; 39 (10): 1902–1910. doi: 10.1161/ATVBAHA.119.311574

7. Otsuka F., Sakakura K., Yahagi K., Joner M., Virmani R. Has our understanding of calcification in human coronary atherosclerosis progressed? Arterioscler. Thromb. Vasc. Biol. 2014; 34 (4): 724–736. doi: 10.1161/ATVBAHA.113.302642

8. Bäck M., Aranyi T., Cancela M.L., Carracedo M., Conceição N., Leftheriotis G., Macrae V., Martin L., Nitschke Y., Pasch A., Quaglino D., Rutsch F., Shanahan C., Sorribas V., Szeri F., Valdivielso P., Vanakker O., Kempf H. Endogenous calcification inhibitors in the prevention of vascular calcification: A consensus statement from the COST action EuroSoftCalcNet. Front. Cardiovasc. Med. 2019; 5: 196. doi: 10.3389/fcvm.2018.00196

9. Roijers R.B., Debernardi N., Cleutjens J.P., Schurgers L.J., Mutsaers P.H., van der Vusse G.J. Microcalcifications in early intimal lesions of atherosclerotic human coronary arteries. Am. J. Pathol. 2011; 178 (6): 2879–2887. doi: 10.1016/j.ajpath.2011.02.004

10. Carino A., Ludwig C., Cervellino A., Müller E., Testino A. Formation and transformation of calcium phosphate phases under biologically relevant conditions: Experiments and modelling. Acta Biomater. 2018; 74: 478–488. doi: 10.1016/j.actbio.2018.05.027

11. Chen J., Peacock J.R., Branch J., Merryman W.D. Biophysical analysis of dystrophic and osteogenic models of valvular calcification. J. Biomech. Eng. 2015; 137 (2): 020903. doi: 10.1115/1.4029115

12. Hutcheson J.D., Goettsch C., Bertazzo S., Maldonado N., Ruiz J.L., Goh W., Yabusaki K., Faits T., Bouten C., Franck G., Quillard T., Libby P., Aikawa M., Weinbaum S., Aikawa E. Genesis and growth of extracellular-vesicle-derived microcalcification in atherosclerotic plaques. Nat. Mater. 2016; 15 (3): 335–343. doi: 10.1038/nmat4519

13. Burgmaier M., Milzi A., Dettori R., Burgmaier K., Marx N., Reith S. Co-localization of plaque macrophages with calcification is associated with a more vulnerable plaque phenotype and a greater calcification burden in coronary target segments as determined by OCT. PLoS One. 2018; 13 (10): e0205984. doi: 10.1371/journal.pone.0205984

14. Fuery M.A., Liang L., Kaplan F.S., Mohler E.R 3rd. Vascular ossification: Pathology, mechanisms, and clinical implications. Bone. 2018; 109: 28–34. doi: 10.1016/j.bone.2017.07.006

15. Pettenazzo E., Deiwick M., Thiene G., Molin G., Glasmacher B., Martignago F., Bottio T., Reul H., Valente M. Dynamic in vitro calcification of bioprosthetic porcine valves evidence of apatite crystallization. J. Thorac. Cardiovasc. Surg. 2001; 121 (3): 500–509. doi: 10.1067/mtc.2001.112464

16. Murungi J.I., Thiam S., Tracy R.E., Robinson J.W., Warner I.M. Elemental analysis of soft plaque and calcified plaque deposits from human coronary arteries and aorta. J. Environ. Sci. Health. A. Tox. Hazard. Subst. Environ. Eng. 2004; 39 (6): 1487–1496. doi:10.1081/ese-120037848

17. Balachandran K., Sucosky P., Jo H., Yoganathan A.P. Elevated cyclic stretch induces aortic valve calcification in a bone morphogenic protein-dependent manner. Am. J. Pathol. 2010; 177 (1): 49–57. doi: 10.2353/ajpath.2010.090631

18. Mikroulis D., Mavrilas D., Kapolos J., Koutsoukos P.G., Lolas C. Physicochemical and microscopical study of calcific deposits from natural and bioprosthetic heart valves. Comparison and implications for mineralization mechanism. J. Mater. Sci. Mater. Med. 2002; 13 (9): 885–889. doi: 10.1023/a:1016556514203

19. Cheng C.L., Chang H.H., Huang P.J., Wang W.C., Lin S.Y. Ex vivo assessment of valve thickness/ calcification of patients with calcific aortic stenosis in relation to in vivo clinical outcomes. J. Mech. Behav. Biomed. Mater. 2017; 74: 324–332. doi: 10.1016/j.jmbbm.2017.06.020

20. Cottignoli V., Relucenti M., Agrosì G., Cavarretta E., Familiari G., Salvador L., Maras A. Biological niches within human calcified aortic valves: towards understanding of the pathological biomineralization process. Biomed. Res. Int. 2015; 2015: 542687. doi: 10.1155/2015/542687

21. Mangialardo S., Cottignoli V., Cavarretta E., Salvador L., Postorino P., Maras A. Pathological biominerals: raman and infrared studies of bioapatite deposits in human heart valves. Appl. Spectrosc. 2012; 66 (10): 1121–1127. doi: 10.1366/12-06606

22. Cottignoli V., Cavarretta E., Salvador L., Valfré C., Maras A. Morphological and chemical study of pathological deposits in human aortic and mitral valve stenosis: a biomineralogical contribution. Patholog. Res. Int. 2015; 2015: 342984. doi: 10.1155/2015/342984