Raman microspectroscopy of human aortic valves: investigation of the local and global biochemical changes associated with calcification in aortic stenosis

Abstract

Raman microspectroscopy was applied to characterize the local and global biochemical changes associated with calcification in human stenotic aortic valves.

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

“…Glucose supplementation in the in OSM medium, even in the high concentration (4.5 g/L), did not cause significant changes. Nevertheless, the calcification in the form of inorganic phosphate salt deposits that were found in aortic valve tissue was not observed in HAVICs in in vitro cell culture upon incubation with OSM and OSM/glucose for 7 days. Previous studies highlight the extracellular origin of calcification process during which nucleation of calcium nodules takes place in ECM rich in type I collagen and fibronectin .…”

“…Its effect was also tested on vascular SMCs, proving the induction of osteogenic morphology, physiology, and gene expression, which may be a contributing factor to the formation of calcifying vascular cells . Moreover, we have previously found that lipid deposits accompany calcification in the tissue of stenotic aortic valves …”

“…Cellular models enable to reflect of various physiological and pathological processes. In continuation to our previous studies on calcification in the stenotic aortic valves, we investigated the chemical alterations arising from induced calcification in isolated HAVICs from patients with AS for the first time using confocal Raman imaging. To evoke calcification, we applied standard OSM.…”

“…Therefore, alternative methods of chemical characterization of calcification are necessary, and one of such is Raman microscopy that is an unbiased, label‐free technique providing information about distribution of components in the studied sample with the subcellular resolution. We have previously demonstrated that confocal Raman microscopy might be a valuable tool to characterize size and content of calcified deposits in stenotic aortic valves . We have shown that depending on the size of deposits, their chemical composition significantly differs; mineralization grains defined as small (<0.7 μm in diameter), medium (0.7–1.6 μm in diameter), and large (>1.6 μm in diameter) contained mostly octacalcium phosphate‐like compound, tricalcium phosphate, mixed with the salt containing the acidic phosphate groups (HPO 4 2− ) and stable B‐type hydroxyapatite, respectively .…”

“…Although we interpreted this finding as a change of the chemical composition accompanying maturation and growth of calcified deposits, different composition of grains might be alternatively linked to different mechanisms of calcification. Additionally, Raman images of calcified tissue have clearly exhibited lipid aggregation in the close vicinity of mineralization and enabled for determination of lipids' composition (mainly cholesterol and its esters) and, in some cases, carotenoids that co‐localized with the identified lipid moieties . At the same time, local lipid aggregation around calcified tissue did not globally result in the increase of the lipid level, contrarily, the increase of the protein content, most probably due to fibrosis, was detected in stenotic valves …”