ELECTRON MICROSCOPY-ADAPTED PHTHALOCYANINE AND VON KOSSA HISTOCHEMISTRY: A POWERFUL TOOL TO STUDY AORTIC VALVE CALCIFICATION IN “AD HOC” MODELS AND ACTUAL DISEASE

Valve calcification is a multifactorial disorder whose etiopathogenesis remains largely unclear. In our laboratory, the use of copper phthalocyanine Cuprolinic Blue (CB) for histochemical reactions under electron microscopy has provided deeper insights into the pro-calcific degeneration involving aortic valve interstitial cells (VICs) in ad hoc in vivo and in vitro calcification models.1-4 Specifically, CB dissolved in acidulated buffer, gently solubilizing hydroxyapatite (HA) crystals, promotes simultaneous unmasking and staining of acidic lipid material (PPM) derived from organelle and plasma membrane breakdown in VICs, alongside nuclear chromatin and ribosomal derivatives. A major pro-calcific role is clearly exhibited by PPM-derived CB-positive layers (PPLs) forming at VIC edges, as revealed by the selective overlapping of non-removed HA crystals. Blebbing of PPLs produces PPL-lined cell byproducts, including final calcospherulae, which mediate calcification spreading throughout the extracellular spaces. The ability of PPLs to act as major HA nucleators is further confirmed by superimposition of metallic silver particles after post-embedding von Kossa reaction on semithin sections and their re-embedding. This VIC degeneration is also paralleled by overexpression of cytosolic phospholipase A2α (cPLA2α).5 Importantly, these findings are now being demonstrated to occur also in true calcific aortic valve disease (CAVD), challenging the currently prevailing paradigm that attributes valve calcification primarily to osteogenic processes. In conclusion, the visualization protocols applied both in suitable pro-calcific models, including future specifically designed co-cultures, and in actual CAVD, along with further investigations into cPLA2α involvement, will contribute valuable insights into ectopic calcification. 1. Ortolani F, et al. Connect Tissue Res 2002;43:44-55. 2. Ortolani F, et al. Histol Histopathol 2003;18:1131-40. 3. Ortolani F, et al. Histol Histopathol 2007;22:261-72. 4. Bonetti A, et al. J Histochem Cytochem 2017;65:125-38. 5. Bonetti A, et al. Int J Mol Sci 2020;21:6398.