Carotenoids co-localize with hydroxyapatite, cholesterol, and other lipids in calcified stenotic aortic valves. Ex vivo Raman maps compared to histological patterns

Abstract

Unlike its application for atherosclerotic plaque analysis, Raman microspectroscopy was sporadically used to check the sole nature of bioapatite deposits in stenotic aortic valves, neglecting the involvement of accumulated lipids/lipoproteins in the calcific process. Here, Raman microspectroscopy was employed for examination of stenotic aortic valve leaflets to add information on nature and distribution of accumulated lipids and their correlation with mineralization in the light of its potential precocious diagnostic use. Cryosections from surgically explanted stenotic aortic valves (n=4) were studied matching Raman maps against specific histological patterns. Raman maps revealed the presence of phospholipids/triglycerides and cholesterol, which showed spatial overlapping with one another and Raman-identified hydroxyapatite. Moreover, the Raman patterns correlated with those displayed by both von-Kossa-calcium- and Nile-blue-stained serial cryosections. Raman analysis also provided the first identification of carotenoids, which co-localized with the identified lipid moieties. Additional fit concerned the distribution of collagen and elastin. The good correlation of Raman maps with high-affinity staining patterns proved that Raman microspectroscopy is a reliable tool in evaluating calcification degree, alteration/displacement of extracellular matrix components, and accumulation rate of different lipid forms in calcified heart valves. In addition, the novel identification of carotenoids supports the concept that valve stenosis is an atherosclerosis-like valve lesion, consistently with their previous Raman microspectroscopical identification inside atherosclerotic plaques.

Raman imaging of macrophages incubated with triglyceride-enriched oxLDL visualizes translocation of lipids between endocytic vesicles and lipid droplets

“…The intense Raman bands of -carotene were used for the investigation of potential drug-delivery applications of polymeric nanoparticles (43). In another study, -carotene was found colocalized with lipid moieties in plaque depositions of aortic valves (44). The authors assumed that -carotene derived from LDLs taken up by the macrophages in the arterial wall, a concept that is supported by our results.…”

Assessment of Unicuspid Aortic Valve Stenosis Using Multimodality Imaging, X-ray Radiography and Raman Analysis

“…In addition, small Raman peaks at 1161 and 15121 516 cm −1 were found in all Raman spectra, which corresponded to the β-carotene. 27,28) From the Raman spectra in Figure 5, the calcified UAV leaflet of this AS patient was mainly constructed by uneven random distribution of 3 portions: a calcium hydroxyapatite-rich part (55.8%), calcium hydroxyapatite and lipid part (15.4%), and lipid-rich part (28.8%). The calcium hydroxyapatite-rich part was randomly distributed over 50% on the surface of UAV, confirming that this was a unicommissural UAV with severe calcification.…”

Is there still room for novelty, in histochemical papers?

“…111-113 Imaging techniques were the subject of some articles. 114-116 Raman microspectroscopy was used to investigate stenotic aortic valve leaflets to get information on the composition and distribution of accumulated lipids, in the attempt to correlate their presence with mineralization as a precocious diagnostic marker. 114 Magnetic resonance imaging (MRI) was used to perform a longitudinal study in vivo on the spine changes occurring in an experimental rat model of ankylosing spondylitis, and the obtained results were validated at the end of the experiments by micro-computerized tomography and histological examination: this non-invasive approach may potentially be applied to follow the progress of this disease and plan therapeutic interventions in humans.…”

“…114-116 Raman microspectroscopy was used to investigate stenotic aortic valve leaflets to get information on the composition and distribution of accumulated lipids, in the attempt to correlate their presence with mineralization as a precocious diagnostic marker. 114 Magnetic resonance imaging (MRI) was used to perform a longitudinal study in vivo on the spine changes occurring in an experimental rat model of ankylosing spondylitis, and the obtained results were validated at the end of the experiments by micro-computerized tomography and histological examination: this non-invasive approach may potentially be applied to follow the progress of this disease and plan therapeutic interventions in humans. 115 Advanced MRI techniques, such as MRI microscopy, Magnetic Resonance Spectroscopy, functional MRI, and Diffusion Tensor Imaging may be envisaged as suitable minimally-invasive techniques to investigate in vivo not only the morphological features of living organs and tissues, but also their functional, biophysical and molecular characteristics.…”