Phospholipids accumulation and calcification in cultured primary human aortic valve interstitial cells: New insights revealed by confocal Raman imaging

Abstract

Raman imaging was applied to investigate calcification process in situ in primary human aortic valve interstitial cells (HAVICs) isolated from patients with developed non-rheumatic aortic stenosis (AS). To invoke calcification, cells were incubated in an osteogenic medium (OSM) in some cases supplemented with glucose. Surprisingly, upon 7 days of incubation, no calcification in the form of inorganic salts was detected, instead, the increase of lipid inclusions containing phospholipids inside HAVICs was observed, probably in the sites involved in further mineral precipitation. Glucose supplementation (reflecting diabetes mellitus influence on AS) slightly decreased the overall content of phospholipids. A long incubation time resulted in the formation of significant amounts of inorganic calcium salts extracellularly and only in the presence of type I collagen. In the absence of type I collagen, calcium oxalate dihydrate was observed inside the cells. Due to application of Raman imaging, an unbiased, label-free method of high-spatial resolution and chemical specificity, it was possible to demonstrate unequivocally that calcification in the form of hydroxyapatite took place extracellularly in the presence of type I collagen and required long incubation with OSM.