Raman imaging-based phenotyping of murine primary endothelial cells to identify disease-associated biochemical alterations.

Raman spectroscopy is successfully becoming an analytical tool used to characterize alterations in the biochemical composition of cells. In this work, we identify the features of Raman spectra of murine primary endothelial cells (EC) isolated from lungs, heart, liver, brain, kidney and aorta of normal mice, as well as from heart, lung and liver in a murine model of heart failure (HF) in Tgαq*44 mice. Primary cells were measured in suspension immediately after their isolation. Raman images showed that isolated primary EC were elliptical or circular, and did not show organ-specific spectral features for any of the studied organ, i.e. lungs, heart, liver, brain, kidney and aorta. Principal Component Analysis pairwise analysis of primary endothelial cells from FVB mice and Tgαq*44 mice revealed an increased protein content in EC isolated from the heart and increased lipid content in EC isolated from the lung in Tgαq*44 mice. No significant differences were found in the EC isolated from the liver using the same chemometric procedure. To our knowledge, this is the first report in which Raman spectroscopy has been used to characterize the biochemical phenotype of primary murine EC with developing HF. This pilot study shows that Raman-based analysis of freshly isolated primary EC did not revealed organ-specific features, however disease-associated changes were found in the coronary and pulmonary EC in the early stage of heart failure in Tgαq*44 mice.

Labeled vs. Label-Free Raman Imaging of Lipids in Endothelial Cells of Various Origins.

Endothelial cells (EC) constitute a single layer of the lining of blood vessels and play an important role in maintaining cardiovascular homeostasis. Endothelial dysfunction has been recognized as a primary or secondary cause of many diseases and it manifests itself, among others, by increased lipid content or a change in the lipid composition in the EC. Therefore, the analysis of cellular lipids is crucial to understand the mechanisms of disease development. Tumor necrosis factor alpha (TNF-α)-induced inflammation of EC alters the lipid content of cells, which can be detected by Raman spectroscopy. By default, lipid detection is carried out in a label-free manner, and these compounds are recognized based on their spectral profile characteristics. We consider (3S,3'S)-astaxanthin (AXT), a natural dye with a characteristic resonance spectrum, as a new Raman probe for the detection of lipids in the EC of various vascular beds, i.e., the aorta, brain and heart. AXT colocalizes with lipids in cells, enabling imaging of lipid-rich cellular components in a time-dependent manner using laser power 10 times lower than that commonly used to measure biological samples. The results show that AXT can be used to study lipids distribution in EC at various locations, suggesting its use as a universal probe for studying cellular lipids using Raman spectroscopy. The use of labeled Raman imaging of lipids in the EC of various organs could contribute to their easier identification and to a better understanding of the development and progression of various vascular diseases, and it could also potentially improve their diagnosis and treatment.

Raman Optical Activity and Raman spectroscopy of carbohydrates in solution.

This comprehensive study on selected 14 carbohydrates in water solution is an extension of previously published one focused only on solid state analysis. Here, Raman spectroscopy was used as a dedicated method for analysis of carbohydrates in solution, both using a normal effect (RS) and its chiral analogue: Raman Optical Activity spectroscopy (ROA). The compounds were selected as biologically important and representative of all groups: monosaccharides, disaccharides, trisaccharides, cyclodextrines and polysaccharides. RS and ROA spectra are presented together with an expanded discussion on various structures and conformations of studied carbohydrates in the solution taking into account particular regions, i.e. (1) low wavenumber region (250-600 cm-1), (2) anomeric region (600-950 cm-1), (3) fingerprint region (950-1200 cm-1) and (4) CH2and COH deformations region (1200-1500 cm-1). So, the following information can be obtained about: (1) the absolute configuration of the anomeric centre; (2) the configuration of the anomeric centre and the orientation of the anomeric hydroxyl group; (3) the ring structures and the relative orientation of substituents and (4) the conformation of the exocyclic CH2OH (4), respectively. Raman spectroscopy and Raman Optical Activity were shown as unique tools to study complex structures of carbohydrates.

Raman spectroscopic features of primary cardiac microvascular endothelial cells (CMECs) isolated from the murine heart.

Gaining knowledge on the biochemical profile of primary endothelial cells on a subcellular level can contribute to better understanding of cardiovascular disease. In this work, primary cardiac microvascular endothelial cells (CMECs) isolated from the mouse heart and murine H5V endothelial cell line were characterized with the use of a Raman imaging technique. Primary CMECs displayed a distinct Raman-based biochemical phenotype as compared with other cells isolated from the heart and were characterized by a low lipid content. In contrast to the murine H5V endothelial cell line, CMECs did not display lipid droplets (LDs) in the cytoplasm, while the former have many low-unsaturated LDs. In conclusion, Raman imaging is a fast and efficient tool to analyse single coronary endothelial cells in a non-invasive manner that can prove useful to characterize biochemical changes in a single isolated primary endothelial cell from a diseased heart.

Uptake of fatty acids by a single endothelial cell investigated by Raman spectroscopy supported by AFM.

In this work, confocal Raman imaging was used to study the formation of lipid droplets (LDs) in vitro in a single endothelial cell upon incubation with polyunsaturated fatty acids (10 or 25 µM) including arachidonic acid (AA) and its deuterated analog (AA-d8), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Based on the Raman spectra obtained from a single endothelial cell, it was possible to investigate biochemical changes induced by addition of polyunsaturated fatty acids. In particular, the content of lipids in the formed LDs and the unsaturation degree were identified by Raman spectroscopy by marker bands at 1660 cm-1 due to the C[double bond, length as m-dash]C stretching and at ∼3015 cm-1 due to the stretching mode of [double bond, length as m-dash]C-H associated with C[double bond, length as m-dash]C double bonds (except for a deuterated form where these bands are shifted respectively). To establish if the exogenous fatty acid was taken up by the cell and stored in LDs, a deuterium labelled polyunsaturated fatty acid was used. AA-d8 shows characteristic bands at around 2200-2300 cm-1 assigned to the [double bond, length as m-dash]C-D stretching modes. We established the uptake of AA and the accumulation of EPA into newly formed LDs in the endothelial cells. In contrast, no accumulation of DHA in LDs was observed even though LDs were formed upon DHA incubation. Furthermore, using AFM we demonstrated that the presence of LDs in the endothelium affected endothelial stiffness which could have pathophysiological significance. In summary, the results suggest that the formation of LDs in the endothelium involves exogenous and endogenous polyunsaturated fatty acids, and their relative contribution to the LD formation seems distinct for AA, EPA and DHA.