Elastin and collagen interact with cell-derived acidic phospholipid membranes in the progression of mineralization in calcifying aortic valves

INTRODUCTION Elastin fibers have been reported to be prone to mineralization in ageing and in a number of cardiovascular diseases including bioprosthetic valve calcification. Different mechanisms seem to exist which direct these mineralization processes such as in aorta atherosclerotic plaques, where calcification is preceded by accumulation of cholesterol esters and neutral lipids inside altered elastin fibers [1], or that in subdermally implanted aorta wall segments, where calcification occurs at the outer aspect of elastin fibers interacting with undefined amorphous material [2]. Recently, we found that fixation/reactions with glutaraldehyde and cationic copper-phthalocyanine Cuprolinic blue (GACB) at low salt-critical-electrolyte-concentration and pH were suitable for studying calcification in subdermally implanted aortic valves, because of tissue unmasking form mineral and simultaneous visualization at the ultrastructural level of unique, electrondense layers outlining calcifying cells and matrix-vesicle-like structures [3,4]. The observed reactivity and susceptibility to extraction with chloroform-methanol suggested these pericellular layers to be formed by acidic phospholipids which will cluster at cell surfaces replacing cell plasma membranes. Here, a modified glutaraldehyde-malachite-green method (GA-MG) was used to support the concept of lipid involvement in valve calcification and to assess how elastin fibers and collagen fibrils are involved in the mineralization progression from the cells into the extracellular matrix. MATERIAL AND METHODS Animal model inducing calcification: 6-week long implantation of porcine aortic valve leaflets in rat subcutis [5]. Processing of explanted samples: (A) immersion in 25mM sodium acetate buffer, containing 0.05% Cuprolinic blue + 0.05M MgCl2 + 2,5% glutaraldehyde, pH 4.8, at room temperature for 4 days (GACB); (B) immersion in 0.067 mol/L cacodylate buffer solution, containing 3% glutaraldehyde and 0.1% Malachite green, pH 4.8, at 4°C for 18h (GAMG). Post-fixation in 2% OsO4 in phosphate buffer, pH 7.2; dehydration in graded ethanols; embedding in Araldite/Epon. (A-C) thin section staining with uranyl acetate and lead citrate. RESULTS On thin sections, GAMG-subjected samples showed reactivity patterns superimposable to those observed for GACB-treated samples. Namely, decalcifying effect occurred with associated unmasking of cells and matrix vesicles, which appeared to be outlined by MG-reactive, electrondense borders (Fig.1). In addition, mineralization spreading from cells to adjacent extracellular matrix was characterized by outward growing of the MG-reactive material, which enveloped electron-lucent elastin fibers and collagen fibrils, and the additional presence of amorphous material embedding all these components (Fig.2). Despite demineralization, calcium precipitates were observed to be retained by most elastin fibers interacting with MG-reactive material , whereas no mineral deposits was found either on iuxta-cellular fibers still not involved in such a relationship, or those lying in uncalcified extracellular matrix. DISCUSSION GA-MG method reveals acidic phospholipid distribution because it allows lipid retention in the sample, with subsequent formation of electron-dense GA-MG-OsO4-lipid complexes during standard post-fixation with osmium tetroxyde [6]. The lower pH here used allowed to obtain tissue demineralization with preservation of proper reactivness. In the experimental conditions adopted, acidic phospholipids appeared to be accumulated at cell and matrix vesicle surfaces, i.e. where primary calcification occurs, and to be also involved in subsequent mineralization progression into the ECM. Here, elastic fibers and collagen fibrils seem to require prior surface interaction with cell-derived acidic phospholipids before undergoing mineralization, in agreement with the concept that their calcification need interaction with additional molecules [1,3] and/or cell degradation products [3,7]. The concept is supported that elastin fibers can undergo calcification according to different mechanisms which include intrinsic alterations as well as interaction with polyanionic molecules such as proteoglycans, as suggested for pseudoxanthoma elasticum [8], or acidic phospholipids, as suggested by the present data. ACKNOWLEDGEMENTS This work was supported by a special grant by Cassa di Risparmio di Padova e Rovigo Foundation. REFERENCES [1] Bobryshev YV, Lord RS. Atherosclerosis 42, 197-198 (1999). [2] Paule WJ, Bernick S, Strates B, Nimni ME. J. Biomed. Mater. Res. 26, 1169-1177 (1992). [3] Ortolani F, Petrelli L, Tubaro F, Spina M, Marchini M. Connect. Tiss. Res. 43, 44-55 (2002). [4] Ortolani F, Tubaro F, Petrelli L, Gandaglia A, Spina M, Marchini M. Histochem. J. 34, 41-50 (2002). [5] Schoen FJ, Tsao JW, Levy RJ. Am. J. Pathol. 123, 134-145(1986). [6] Bonucci E, Silvestrini G. Bone 15, 153-160 (1994). [7] Kim KM. Scan. Electron. Microsc. 9, 1137-1175 (1995). [8] Pasquali Ronchetti I, Baccarani-Contri M, Fornieri C., Mori G., Quaglino D. jr. Micron 24, 75-89 (1993).