Functional deterioration of vascular mitochondrial and glycolytic capacity in the aortic rings of aged mice.

Vascular ageing is associated with increased arterial stiffness and cardiovascular mortality that might be linked to altered vascular energy metabolism. The aim of this study was to establish a Seahorse XFe96 Analyzer-based methodology for the reliable, functional assessment of mitochondrial respiration and glycolysis in single murine aortic rings and to validate this functional assay by characterising alterations in vascular energy metabolism in aged mice. Healthy young and old C57BL/6 mice were used for the analyses. An optimised setup consisting of the Seahorse XFe96 Analyzer and Seahorse Spheroid Microplates was applied for the mitochondrial stress test and the glycolysis stress test on the isolated murine aortic rings, supplemented with analysis of NAD content in the aorta. To confirm the age-dependent stiffness of the vasculature, pulse wave velocity was measured in vivo. In addition, the activity of vascular nitric oxide synthase and vascular wall morphology were analysed ex vivo. The vascular ageing phenotype in old mice was confirmed by increased aortic stiffness, vascular wall remodelling, and nitric oxide synthase activity impairment. The rings of the aorta taken from old mice showed changes in vascular energy metabolism, including impaired spare respiratory capacity, maximal respiration, glycolysis, and glycolytic capacity, as well as a fall in the NAD pool. In conclusion, optimised Seahorse XFe96-based analysis to study energy metabolism in single aortic rings of murine aorta revealed a robust impairment of functional vascular respiratory and glycolytic capacity in old mice linked to NAD deficiency that coincided with age-related aortic wall remodelling and stiffness.

Uptake and anti-inflammatory effects of liposomal astaxanthin on endothelial cells tracked by Raman and fluorescence imaging.

Astaxanthin (AXT) is a lipophilic antioxidant and anti-inflammatory natural pigment whose cellular uptake and bioavailability could be improved via liposomal encapsulation. Endothelial cells (EC) line the lumen of all blood vessels and are tasked with multiple roles toward maintaining cardiovascular homeostasis. Endothelial dysfunction is linked to the development of many diseases and is closely interconnected with oxidative stress and vascular inflammation. The uptake of free and liposomal AXT into EC was investigated using Raman and fluorescence microscopies. AXT was either encapsulated in neutral or cationic liposomes. Enhanced uptake and anti-inflammatory effects of liposomal AXT were observed. The anti-inflammatory effects of liposomal AXT were especially prominent in reducing EC lipid unsaturation, lowering numbers of lipid droplets (LDs), and decreasing intercellular adhesion molecule 1 (ICAM-1) overexpression, which is considered a well-known marker for endothelial inflammation. These findings highlight the benefits of AXT liposomal encapsulation on EC and the applicability of Raman imaging to investigate such effects.

EdU sensing: The Raman way of following endothelial cell proliferation in vitro and ex vivo.

Endothelial cells line the lumen of all vessels in the body and maintain vascular homeostasis. In particular, endothelial cell regeneration in response to insult sustain functional endothelial layer. EdU (5-ethynyl-2'-deoxyuridine) is an alkyne-tagged proliferation probe that incorporates into newly synthesized DNA and is used for fluorescence imaging of cell proliferation with the use of "click chemistry" reaction with a fluorescent azide. Here, we utilized EdU as a click-free Raman probe for tracking endothelial cell proliferation. Raman imaging of EdU was performed in live endothelial cells, showing an advantage over fluorescence imaging of EdU, as this technique did not require sample fixation and permeabilization. To validate Raman-based imaging of EdU to study endothelial cell proliferation, we showed that when endothelial cells were treated with cycloheximide or doxorubicin to impair the proliferation of endothelial cells, the Raman-based signal of EdU was diminished. Furthermore, endothelial cells proliferation detected using EdU-labelled Raman imaging was compared with fluorescence imaging. Finally, the method of Raman-based EdU imaging was used in the isolated murine aorta ex vivo. Altogether, our results show that Raman-based imaging of EdU provides a novel alternative for fluorescence-based assay to assess endothelial proliferation and regeneration.

Toward Raman Subcellular Imaging of Endothelial Dysfunction.

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.

3D-Printed Soft Lithography for Complex Compartmentalized Microfluidic Neural Devices.

Compartmentalized microfluidic platforms are an invaluable tool in neuroscience research. However, harnessing the full potential of this technology remains hindered by the lack of a simple fabrication approach for the creation of intricate device architectures with high-aspect ratio features. Here, a hybrid additive manufacturing approach is presented for the fabrication of open-well compartmentalized neural devices that provides larger freedom of device design, removes the need for manual postprocessing, and allows an increase in the biocompatibility of the system. Suitability of the method for multimaterial integration allows to tailor the device architecture for the long-term maintenance of healthy human stem-cell derived neurons and astrocytes, spanning at least 40 days. Leveraging fast-prototyping capabilities at both micro and macroscale, a proof-of-principle human in vitro model of the nigrostriatal pathway is created. By presenting a route for novel materials and unique architectures in microfluidic systems, the method provides new possibilities in biological research beyond neuroscience applications.

Detection of 1,2-hydride shifts in the formation of euph-7-ene by the squalene-tetrahymanol cyclase of Tetrahymena pyriformis.

Incubation of samples of 2,3-dihydrosqualene, specifically labeled with deuterium at either carbon position 7 or 11, with an enzyme extract from Tetrahymena pyriformis, containing a squalene-tetrahymanol cyclase, provided specimens of euph-7-enes displaying deuterium patterns consistent with the biosynthetic operation of two consecutive 1,2-hydride shifts.