Multiple diseases are at some point associated with altered endothelial function, and endothelial dysfunction (ED) contributes to their pathophysiology. Biochemical changes of the dysfunctional endothelium are linked to various cellular organelles, including the mitochondria, endoplasmic reticulum, and nucleus, so organelle-specific insight is needed for better understanding of endothelial pathobiology. Raman imaging, which combines chemical specificity with microscopic resolution, has proved to be useful in detecting biochemical changes in ED at the cellular level. However, the detection of spectroscopic markers associated with specific cell organelles, while desirable, cannot easily be achieved by Raman imaging without labeling. This critical review summarizes the current advances in Raman-based analysis of ED, with a focus on a new approach involving molecular Raman reporters that could facilitate the study of biochemical changes in cellular organelles. Finally, imaging techniques based on both conventional spontaneous Raman scattering and the emerging technique of stimulated Raman scattering are discussed.
Here we report a new Raman probe for cellular studies on lipids detection and distribution. It is (3S, 3'S)-astaxanthin (AXT), a natural xanthophyll of hydrophobic properties and high solubility in lipids. It contains a chromophore group, a long polyene chain of eleven conjugated C=C bonds including two in the terminal rings, absorbing light in the visible range that coincides with the excitation of lasers commonly used in Raman spectroscopy for studying of biological samples. Depending on the laser, resonance (excitation in the visible range) or pre-resonance (the near infrared range) Raman spectrum of astaxanthin is dominated by bands at ca. 1008, 1158, and 1520 cm−1 that now can be also a marker of lipids distribution in the cells. We showed that AXT accumulates in lipidic structures of endothelial cells in time-dependent manner that provides possibility to visualize e.g. endoplasmic reticulum, as well as nuclear envelope. As a non-toxic reporter, it has a potential in the future studies on e.g. nucleus membranes damage in live cells in a very short measuring time.
Raman imaging using molecular reporters is a relatively new approach in subcellular investigations. It enables the visualization of organelles in cells with better selectivity and sensitivity compared to the label-free approach. Essentially Raman reporters possess in their structure an alkyne molecular group that can be selectively identified in the spectral region silent for biomolecules, hence facilitate the localization of individual organelles. The aim of this work is to visualize the main cell organelles in endothelial cells (HMEC-1) using established reporters (EdU and MitoBADY), but also to test a new one, namely falcarinol, which exhibits lipophilic properties. Moreover, we tested the possibility to use Raman reporters as a probe to detect changes in distribution of certain organelles after induced endothelial dysfunction (ED) in in vitro models. In both cases, induced ED is characterized by the formation of lipid droplets in the cells, which is why a good tool for the detection of lipid-rich organelles is so important in these studies. Twodimensional Raman images were obtained, visualizing the distribution of selected organic compounds in the cell, such as proteins, lipids, and nucleic acids. Additionally, the distribution of EdU, MitoBADY and falcarinol in endothelial cells was determined. Moreover, we highlight some drawback of established Raman reporter and the need for testing them in various physiological state of the cell.
Small molecules are frequently used as dyes, labels and markers to visualize and probe biophysical processes within cells.
Sickle cell disease (SCD) is a group of disorders that affects hemoglobin due to a mutation of the hemoglobin beta gene (HBB) on chromosome 11. Patients with SCD have atypical hemoglobin molecules called hemoglobinS (HbS), which distort erythrocytes into a “sickle-shape”. Typical symptoms of SCD include periodic episodes of pain, repeated infections, and anemia. This disorder is abundant in sub-Saharan African countries, the Mediterranean region, and also appears in some southern provinces in Turkey. Because of the high concentration of HbS in patients, a high risk of chronic anemia and vaso-occlusive events, such as stroke may deteriorate suddenly. In these conditions, transfusion of blood, especially erythrocytes, can be life-saving. However, chronic blood transfusions may lead to iron overload in SCD patients. Erythrocyte transfusion is associated with a higher risk in most patients with SCD than in the general population. Therefore, chelation therapy has become an important component of the transfusion program to prevent complications of iron accumulation in organs such as liver and heart. In this study, we sought to conduct a systematic review to assess the safety of iron chelating agents used by SCD patients with iron overload mainly due to necessary blood transfusion regime. Our evaluation revealed that in general iron chelation therapy, either deferasirox, deferoxamine or deferiprone, remains the most effective and safest available method to treat iron overload in SCD. Furthermore, current reports do not reflect any significant safety concerns against the use of available chelators.
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