Liver sinusoidal endothelial cells rely on oxidative phosphorylation but avoid processing long-chain fatty acids in their mitochondria.

BACKGROUND: It is generally accepted that endothelial cells (ECs), primarily rely on glycolysis for ATP production, despite having functional mitochondria. However, it is also known that ECs are heterogeneous, and their phenotypic features depend on the vascular bed. Emerging evidence suggests that liver sinusoidal ECs (LSECs), located in the metabolically rich environment of the liver, show high metabolic plasticity. However, the substrate preference for energy metabolism in LSECs remains unclear. METHODS: Investigations were conducted in primary murine LSECs in vitro using the Seahorse XF technique for functional bioenergetic assays, untargeted mass spectrometry-based proteomics to analyse the LSEC proteome involved in energy metabolism pathways, liquid chromatography-tandem mass spectrometry-based analysis of acyl-carnitine species and Raman spectroscopy imaging to track intracellular palmitic acid. RESULTS: This study comprehensively characterized the energy metabolism of LSECs, which were found to depend on oxidative phosphorylation, efficiently fuelled by glucose-derived pyruvate, short- and medium-chain fatty acids and glutamine. Furthermore, despite its high availability, palmitic acid was not directly oxidized in LSEC mitochondria, as evidenced by the acylcarnitine profile and etomoxir's lack of effect on oxygen consumption. However, together with L-carnitine, palmitic acid supported mitochondrial respiration, which is compatible with the chain-shortening role of peroxisomal ß-oxidation of long-chain fatty acids before further degradation and energy generation in mitochondria. CONCLUSIONS: LSECs show a unique bioenergetic profile of highly metabolically plastic ECs adapted to the liver environment. The functional reliance of LSECs on oxidative phosphorylation, which is not a typical feature of ECs, remains to be determined.

Vascular lipid droplets formed in response to TNF, hypoxia, or OA: biochemical composition and prostacyclin generation.

Biogenesis of lipid droplets (LDs) in various cells plays an important role in various physiological and pathological processes. However, the function of LDs in endothelial physiology and pathology is not well understood. In the present work, we investigated the formation of LDs and prostacyclin (PGI2) generation in the vascular tissue of isolated murine aortas following activation by proinflammatory factors: tumor necrosis factor (TNF), lipopolysaccharides (LPS), angiotensin II (AngII), hypoxic conditions, or oleic acid (OA). The abundance, size, and biochemical composition of LDs were characterized based on Raman spectroscopy and fluorescence imaging. We found that blockade of lipolysis by the adipose triglyceride lipase (ATGL) delayed LDs degradation and simultaneously blunted PGI2 generation in aorta treated with all tested proinflammatory stimuli. Furthermore, the analysis of Raman spectra of LDs in the isolated vessels stimulated by TNF, LPS, AngII, or hypoxia uncovered that these LDs were all rich in highly unsaturated lipids and had a negligible content of phospholipids and cholesterols. Additionally, by comparing the Raman signature of endothelial LDs under hypoxic or OA-overload conditions in the presence or absence of ATGL inhibitor, atglistatin (Atgl), we show that Atgl does not affect the biochemical composition of LDs. Altogether, independent of whether LDs were induced by pro-inflammatory stimuli, hypoxia, or OA and of whether they were composed of highly unsaturated or less unsaturated lipids, we observed LDs formation invariably associated with ATGL-dependent PGI2 generation. In conclusion, vascular LDs formation and ATGL-dependent PGI2 generation represent a universal response to vascular proinflammatory insult.

The distinct phenotype of primary adipocytes and adipocytes derived from stem cells of white adipose tissue as assessed by Raman and fluorescence imaging.

Spectroscopy-based analysis of chemical composition of cells is a tool still scarcely used in biological sciences, although it provides unique information about the cell identity accessible in vivo and in situ. Through time-lapse spectroscopic monitoring of adipogenesis in brown and white adipose tissue-derived stem cells we have demonstrated that considerable chemical and functional changes occur along with cells differentiation and maturation, yet yielding mature adipocytes with a similar chemical composition, independent of the cellular origin (white or brown adipose tissue). However, in essence, these stem cell-derived adipocytes have a markedly different chemical composition compared to mature primary adipocytes. The consequences of this different chemical (and, hence, functional) identity have great importance in the context of selecting a suitable methodology for adipogenesis studies, particularly in obesity-related research.

Rac1 regulates lipid droplets formation, nanomechanical, and nanostructural changes induced by TNF in vascular endothelium in the isolated murine aorta.

Endothelial inflammation is recognized as a critical condition in the development of cardiovascular diseases. TNF-induced inflammation of endothelial cells is linked to the formation of lipid droplets, augmented cortical stiffness, and nanostructural endothelial plasma membrane remodelling, but the insight into the mechanism linking these responses is missing. In the present work, we determined the formation of lipid droplets (LDs), nanomechanical, and nanostructural responses in the model of TNF-activated vascular inflammation in the isolated murine aorta using Raman spectroscopy, fluorescence imaging, atomic force microscopy (AFM), and scanning electron microscopy (SEM). We analysed the possible role of Rac1, a major regulator of cytoskeletal organization, in TNF-induced vascular inflammation. We demonstrated that the formation of LDs, polymerization of F-actin, alterations in cortical stiffness, and nanostructural protuberances in endothelial plasma membrane were mediated by the Rac1. In particular, we revealed a significant role for Rac1 in the regulation of the formation of highly unsaturated LDs formed in response to TNF. Inhibition of Rac1 also downregulated the overexpression of ICAM-1 induced by TNF, supporting the role of Rac1 in vascular inflammation. Altogether, our results demonstrate that LDs formation, an integral component of vascular inflammation, is activated by Rac1 that also regulates nanomechanical and nanostructural alterations linked to vascular inflammation.

Rapid shear stress-dependent ENaC membrane insertion is mediated by the endothelial glycocalyx and the mineralocorticoid receptor.

The contribution of the shear stress-sensitive epithelial Na+ channel (ENaC) to the mechanical properties of the endothelial cell surface under (patho)physiological conditions is unclear. This issue was addressed in in vivo and in vitro models for endothelial dysfunction. Cultured human umbilical vein endothelial cells (HUVEC) were exposed to laminar (LSS) or non-laminar shear stress (NLSS). ENaC membrane insertion was quantified using Quantum-dot-based immunofluorescence staining and the mechanical properties of the cell surface were probed with the Atomic Force Microscope (AFM) in vitro and ex vivo in isolated aortae of C57BL/6 and ApoE/LDLR-/- mice. Flow- and acetylcholine-mediated vasodilation was measured in vivo using magnetic resonance imaging. Acute LSS led to a rapid mineralocorticoid receptor (MR)-dependent membrane insertion of ENaC and subsequent stiffening of the endothelial cortex caused by actin polymerization. Of note, NLSS stress further augmented the cortical stiffness of the cells. These effects strongly depend on the presence of the endothelial glycocalyx (eGC) and could be prevented by functional inhibition of ENaC and MR in vitro endothelial cells and ex vivo endothelial cells derived from C57BL/6, but not ApoE/LDLR-/- vessel. In vivo In C57BL/6 vessels, ENaC- and MR inhibition blunted flow- and acetylcholine-mediated vasodilation, while in the dysfunctional ApoE/LDLR-/- vessels, this effect was absent. In conclusion, under physiological conditions, endothelial ENaC, together with the glycocalyx, was identified as an important shear stress sensor and mediator of endothelium-dependent vasodilation. In contrast, in pathophysiological conditions, ENaC-mediated mechanotransduction and endothelium-dependent vasodilation were lost, contributing to sustained endothelial stiffening and dysfunction.

Lipid Droplets Formation Represents an Integral Component of Endothelial Inflammation Induced by LPS.

Endothelial inflammation is the hallmark of vascular pathology often proceeding with cardiovascular diseases. Here, we adopted a multiparameter approach combining various imaging techniques at the nano- and microscale (Raman, AFM and fluorescence) to investigate endothelial inflammation in response to lipopolysaccharides (LPS) in vitro in human microvascular endothelial cells (HMEC-1) with a focus on lipid droplets (LDs) formation. Our results show that LPS-induced LDs in HMEC-1 have a composition depending on LPS-incubation time and their formation requires the presence of serum. Robust endothelial inflammation induced by LPS was linked to LDs composed of highly unsaturated lipids, as well as prostacyclin release. LPS-induced LDs were spatially associated with nanostructural changes in the cell membrane architecture. In summary, LDs formation represents an integral component of endothelial inflammation induced by LPS.

Estimation of the content of lipids composing endothelial lipid droplets based on Raman imaging.

Lipid droplets (LDs) are dynamic organelles involved in intracellular lipid metabolism, and the biogenesis of LDs in endothelium is triggered by the excess of lipids in the environment. In this paper we present the methodology aimed to define the composition of endothelial LDs formed upon stimulation with oleic acid (OA) in two models: endothelial cells cultured in vitro and in isolated blood vessel ex vivo. The biochemical composition of LDs was determined using Raman imaging, followed by the lipid unsaturation calibration analysis and modelling of spectral bands based on individual spectra of selected lipids. Among LDs formed in response to OA in vitro or ex vivo conditions there were two types of LDs; those with more unsaturated (average number of CC bonds equalled 1.40) or saturated (average number of CC bonds equalled 0.95) lipids. The modelling of endothelial LDs composition revealed the OA represented a major component of LDs (80.6-91.3%) with an important content of arachidonic acid (8.7-19.4%). In conclusion, endothelial LDs consist of exogenous oleic acid uptaken from the extracellular space, and the endogenous arachidonic acid released from plasma membranes.

Heterogeneity of chemical composition of lipid droplets in endothelial inflammation and apoptosis.

Lipid droplets (LDs) play regulatory role in various cells but their significance in endothelial pathophysiology is still not well understood. Here, we studied LDs in in situ endothelial cells (ECs) in isolated blood vessels stimulated with pro-inflammatory or pro-apoptotic stimuli using Raman and fluorescence imaging. Endothelial inflammation induced by murine TNF-α (mTNF-α) was featured by overexpression of ICAM-1, vWF, increased production of PGI2, and was associated with the formation of low number of LDs. However in the presence of atglistatin, the inhibitor of triacyclglycerols hydrolysis, the number of LDs significantly increased. In contrast, in endothelium stimulated by human TNF-α (hTNF-α) or FasL, apart from endothelial inflammation, displayed also apoptosis as evidenced by high annexin expression and significant LDs formation. Raman imaging confirmed that LDs were localized in endothelium and revealed significant heterogeneity in biochemical composition of endothelial LDs that dependent on endothelial stimuli. Repertoire of LDs included LDs rich in highly unsaturated lipids, assigned to the inflammation, as well as LDs featured by more saturated lipids linked to apoptosis, where Raman signals indicating content of cholesterol and phospholipids were higher for endothelial apoptosis in comparison to endothelial inflammation. The heterogeneity in chemical composition of LDs suggested more complex pathophysiological role of endothelial LDs then previously appreciated.

Light Microscopy and Raman Imaging of Carotenoids in Plant Cells In Situ and in Released Carotene Crystals.

Light microscopy with a bright field mode offers an easy and fast examination of plant specimen for carotenoid presence in its cells. Using basic techniques such as hand sectioned or squashed preparations, carotenoid-rich chromoplasts can be identified without applying any staining procedure and their localization within the cell, their shape and number can be assessed. More detailed information can be obtained by using Raman spectroscopy which is suitable for the analysis of carotenoids due to their unique Raman spectra and allows semiquantification of their contents. Raman imaging (mapping) can be additionally used to show the distribution of carotenoids within the sample. Raman spectra can be taken from extracted carotenoids but can be also obtained directly from plant tissues or cells as Raman measurements are nondestructive for the sample. Here we describe preparations of intact tissue samples, monolayer cell samples, isolated protoplasts as well as carotene crystals released from chromoplasts that are suitable for subsequent observations using light microscopy and for analysis using Raman spectroscopy.

Combined Raman- and AFM-based detection of biochemical and nanomechanical features of endothelial dysfunction in aorta isolated from ApoE/LDLR-/- mice.

Endothelial dysfunction is recognized as a critical condition in the development of cardiovascular disorders. This multifactorial process involves changes in the biochemical and mechanical properties of endothelial cells leading to disturbed release of vasoprotective mediators. Hypercholesterolemia and increased stiffness of the endothelial cortex are independently shown to result in reduced release of nitric oxide and thus endothelial dysfunction. However, direct evidence linking these parameters to each other is missing. Here, a novel method combining Raman spectroscopy for biochemical analysis and Atomic Force Microscopy (AFM) for analyzing the endothelial nanomechanics was established. Using this dual approach, the same areas of native ex vivo aortas were investigated, either derived from mice with endothelial dysfunction (ApoE/LDLR-/-) or wild type mice. In particular an increased intracellular lipid content and elevated cortical stiffness/elasticity were shown in ApoE/LDLR-/- aortas, demonstrating a direct link between endothelial dysfunction, the biochemical composition and the nanomechanical properties of endothelial cells.

Raman spectroscopy as a novel tool for fast characterization of the chemical composition of perivascular adipose tissue.

One of the new targets of untapped therapeutic potential is perivascular adipose tissue (pVAT). pVAT releases a plethora of pro- and anti-inflammatory agents and is involved in the inflammatory response of the vascular wall, playing a key role in various cardiovascular pathologies. Both fiber optic Raman spectroscopy with a high-spatial resolution probe and Raman microscopy were applied to study various types of adipose tissue with the emphasis on pVATs of the thoracic and abdominal aorta and the mesenteric artery, as well as epididymal and interscapular adipose tissue for comparison. Our results demonstrated that the lipid unsaturation degree was clearly distinct in various types of adipose tissue and was influenced by the age of animals. In particular, the basal unsaturation level of pVATs of the abdominal aorta and the mesenteric artery was considerably higher than that of the thoracic aorta and a significant increase of the unsaturation level of pVAT with age was observed showing that aging has a considerable impact on the pVAT's chemical composition. Overall, our results show that Raman spectroscopy is a sensitive tool to determine the perivascular adipose tissue chemical composition that appears to be vascular-bed specific.

Rapid diagnostics of liver steatosis by Raman spectroscopy via fiber optic probe: a pilot study.

Raman spectroscopy via fiber optic probes omits some of the major limitations related to ex vivo preparation of tissue samples (e.g. fixation, freezing, cutting) and enables rapid registration of spectra, therefore, this technique has the potential to become a useful diagnostic tool. In this work, we evaluated the applicability of Raman spectroscopy via fiber optic probe for rapid assessment of lipid content in the liver in the context of its potential application as a tool to verify the degree of liver steatosis. Non-alcoholic fatty liver disease (NAFLD) is a common liver disorder that is characterized by excessive lipid accumulation within hepatic tissue and is associated with insulin resistance and metabolic syndrome. Raman spectroscopy via fiber optic probe was applied to investigate the biochemical status of the liver in mild (mice fed a high-fat diet) and severe (db/db mice) models of NAFLD. A considerable increase in lipid content without substantial alterations in composition was observed in mild liver steatosis. In contrast, more severe liver steatosis caused not only a significant lipid content increase, but also hepatic cholesterol accumulation accompanied by significant loss of hepatic vitamin A content. Chemometric analysis based on average Raman spectra recorded via fiber optic probe provided discrimination of mild and severe liver steatosis and control livers with high sensitivity and specificity. In conclusion, our work demonstrates that a relatively simple Raman setup equipped with a commercial fiber optic probe combined with basic chemometric analysis enables rapid quantification of liver steatosis.

Raman, AFM and SNOM high resolution imaging of carotene crystals in a model carrot cell system.

Three non-destructive and complementary techniques, Raman imaging, Atomic Force Microscopy and Scanning Near-field Optical Microscopy were used simultaneously to show for the first time chemical and structural differences of carotenoid crystals. Spectroscopic and microscopic scanning probe measurements were applied to the released crystals or to crystals accumulated in a unique, carotenoids rich callus tissue growing in vitro that is considered as a new model system for plant carotenoid research. Three distinct morphological crystal types of various carotenoid composition were identified, a needle-like, rhomboidal and helical. Raman imaging using 532 and 488 nm excitation lines provided evidence that the needle-like and rhomboidal crystals had similar carotenoid composition and that they were composed mainly of ß-carotene accompanied by α-carotene. However, the presence of α-carotene was not identified in the helical crystals, which had the characteristic spatial structure. AFM measurements of crystals identified by Raman imaging revealed the crystal topography and showed the needle-like and rhomboidal crystals were planar but they differed in all three dimensions. Combining SNOM and Raman imaging enabled indication of carotenoid rich structures and visualised their distribution in the cell. The morphology of identified subcellular structures was characteristic for crystalline, membraneous and tubular chromoplasts that are plant organelles responsible for carotenoid accumulation in cells.

Raman and infrared spectroscopy of carbohydrates: A review.

Carbohydrates are widespread and naturally occurring compounds, and essential constituents for living organisms. They are quite often reported when biological systems are studied and their role is discussed. However surprisingly, up till now there is no database collecting vibrational spectra of carbohydrates and their assignment, as has been done already for other biomolecules. So, this paper serves as a comprehensive review, where for selected 14 carbohydrates in the solid state both FT-Raman and ATR FT-IR spectra were collected and assigned. Carbohydrates can be divided into four chemical groups and in the same way is organized this review. First, the smallest molecules are discussed, i.e. monosaccharides (d-(-)-ribose, 2-deoxy-d-ribose, l-(-)-arabinose, d-(+)-xylose, d-(+)-glucose, d-(+)-galactose and d-(-)-fructose) and disaccharides (d-(+)-sucrose, d-(+)-maltose and d-(+)-lactose), and then more complex ones, i.e. trisaccharides (d-(+)-raffinose) and polysaccharides (amylopectin, amylose, glycogen). Both Raman and IR spectra were collected in the whole spectral range and discussed looking at the specific regions, i.e. region V (3600-3050cm-1), IV (3050-2800cm-1) and II (1200-800cm-1) assigned to the stretching vibrations of the OH, CH/CH2 and C-O/C-C groups, respectively, and region III (1500-1200cm-1) and I (800-100cm-1) dominated by deformational modes of the CH/CH2 and CCO groups, respectively. In spite of the fact that vibrational spectra of saccharides are significantly less specific than spectra of other biomolecules (e.g. lipids or proteins), marker bands of the studied molecules can be identified and correlated with their structure.

Lipids, hemoproteins and carotenoids in alive Rhodotorula mucilaginosa cells under pesticide decomposition - Raman imaging study.

Various species of yeasts are gaining attention as producers of nutraceuticals and biofuels and due to their capacity to biodegrade chemical waste. Rhodotorula mucilaginosa is one of the most oleaginous species of yeast, an efficient de novo carotenoid producer and was reported to be capable of decomposing of organic pesticides. In this work we studied the influence of a toxic pesticide, diazinone, on production of storage (lipids) and protective (carotenoids, hemoproteins) compounds by Rh. mucilaginosa alive cells with the help of Raman imaging. It occurred that the yeast in non-oleaginous phase and aerobic environment was rich in carotenoids and their level increased significantly under incubation with diazinone, while anaerobic environment resulted in production of both carotenoids and hemoproteins and the level of the latter decreased under the influence of the pesticide. For yeasts in oleaginous phase, it was concluded that lipid production (via triggering of NAD+ accumulation and increase of the NO level) resulted in nitrosative stress leading to flavohemoprotein synthesis and was associated with the increase of the mitochondrial activity.

3D Raman imaging of systemic endothelial dysfunction in the murine model of metastatic breast cancer.

It was recently reported in the murine model of metastatic breast cancer (4T1) that tumor progression and development of metastasis is associated with systemic endothelial dysfunction characterized by impaired nitric oxide (NO) production. Using Raman 3D confocal imaging with the analysis of the individual layers of the vascular wall combined with AFM endothelial surface imaging, we demonstrated that metastasis-induced systemic endothelial dysfunction resulted in distinct chemical changes in the endothelium of the aorta. These changes, manifested as a significant increase in the protein content (18%) and a slight decrease in the lipid content (4%), were limited to the endothelium and did not occur in the deeper layers of the vascular wall. The altered lipid to protein ratio in the endothelium, although more pronounced in the fixed vascular wall, was also observed in the freshly isolated unfixed vascular wall samples in the aqueous environment (12 and 7% change of protein and lipid content, respectively). Our results support the finding that the metastasis induces systemic endothelial dysfunction that may contribute to cancer progression.

Raman microscopy as a novel tool to detect endothelial dysfunction.

Raman microscopy, a label-free method with high spatial resolution, shows growing potential in various fields of medical diagnostics. Several proof-of-concept studies related to the application of Raman microscopy to detect endothelial dysfunction are summarized in this work. Both ex vivo measurements of the tissues in the murine models of endothelial pathologies, as well as in vitro investigations of the cell cultures in the context of cellular transport, drug action and inflammation processes are discussed. The future directions in application of Raman spectroscopy-based methods in such studies are also described.

Interplay between carotenoids, hemoproteins and the "life band" origin studied in live Rhodotorula mucilaginosa cells by means of Raman microimaging.

Raman microimaging of live Rhodotorula mucilaginosa cells, cultured under aerobic and anaerobic conditions, showed striking differences in the composition and distribution of cell components. The analysis of these differences and recovery of oxidative phosphorylation upon environmental changes enabled the interrelation of carotenoids, hemoproteins and the unknown species considered as the "Raman signature of life".