Spatially Offset Raman Spectroscopy toward In Vivo Assessment of the Adipose Tissue in Cardiometabolic Pathologies.

Spatially offset Raman spectroscopy (SORS) enhanced the capabilities of Raman spectroscopy for the depth-resolved analysis of biological and diffusely scattering samples. This technique offers selective probing of subsurface layers, providing molecular insights without invasive procedures. While SORS has found application in biomedical research, up to now, studies have focused mainly on the detection of mineralization of bones and tissues. Herein, for the first time, SORS is used to assess the soft, organic tissue beneath the skin's surface. In this study, we demonstrate the diagnostic utility of a hand-held SORS device for evaluating the chemical composition of the adipose tissue. We compared perigonadal white adipose tissue (gWAT) in a murine model of atherosclerosis, heart failure, and high-fat diet (HFD) induced obesity. Our results reveal distinct chemical differences in gWAT between HFD-fed and control mice, showcasing the potential of SORS for intravital adipose tissue phenotype characterization. Furthermore, our findings underscore the effectiveness of SORS as a valuable tool for noninvasive assessment of the adipose tissue composition, holding potential diagnostic significance for metabolic disorders.

Efficient delivery of carotenoids to adipocytes with albumin.

Carotenoids are very effectively delivered by albumin to adipocytes. The uptake of carotenoids to the cells occurs in the form of self-aggregates that localize in the vicinity of the adipocyte membrane, as shown by high spatial resolution Raman spectroscopy. The binding of carotenoids to albumin and the mechanism of their transport were elucidated with the help of chiroptical spectroscopies, in tandem with molecular docking and molecular dynamics simulations. In particular, apart from the recognized high affinity pocket of albumin that binds a carotenoid monomer in domain I, we have identified a hydrophobic periphery area in domain IIIB that loosely bounds the self-aggregated carotenoid in aqueous media and enables its easy detachment in hydrophobic environments. This explains the effectiveness of albumins as nanocarriers of carotenoids to adipocytes in vitro.

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.

What is the ability of inflamed endothelium to uptake exogenous saturated fatty acids? A proof-of-concept study using spontaneous Raman, SRS and CARS microscopy.

Endothelial cells (EC) in vivo buffer and regulate the transfer of plasma fatty acid (FA) to the underlying tissues. We hypothesize that inflammation could alter the functionality of the EC, i.e., their capacity and uptake of different FA. The aim of this work is to verify the functionality of inflamed cells by analyzing their ability to uptake and accumulate exogenous saturated FA. Control and inflammatory human microvascular endothelial cells stimulated in vitro with two deuterium-labeled saturated FA (D-FA), i.e., palmitic (D31-PA) and myristic (D27-MA) acids. Cells were measured both by spontaneous and stimulated Raman imaging to extract detailed information about uptaken FA, whereas coherent anti-Stokes Raman scattering and fluorescence imaging showed the global content of FA in cells. Additionally, we employed atomic force microscopy to obtain a morphological image of the cells. The results indicate that the uptake of D-FA in inflamed cells is dependent on their concentration and type. Cells accumulated D-FA when treated with a low concentration, and the effect was more pronounced for D27-MA, in normal cells, but even more so, in inflamed cells. In the case of D31-PA, a slightly increased uptake was observed for inflamed cells when administered at higher concentration. The results provide a better understanding of the EC inflammation and indicate the impact of the pathological state of the EC on their capacity to buffer fat. All the microscopic methods used showed complementarity in the analysis of FA uptake by EC, but each method recognized this process from a different perspective.

Astaxanthin as a new Raman probe for biosensing of specific subcellular lipidic structures: can we detect lipids in cells under resonance conditions?

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.

A new approach to study human perivascular adipose tissue of the internal mammary artery by fiber-optic Raman spectroscopy supported by spectral modelling.

Perivascular adipose tissue (PVAT) regulates vascular function and represents a novel therapeutic target in vascular diseases. In this work, a new approach based on fiber-optic Raman spectroscopy and spectral modelling was used to characterize the chemical content of the PVAT of the internal mammary artery (IMA) of patients with advanced coronary atherosclerosis (n = 10) undergoing coronary bypass surgery. Our results showed a high degree of lipid unsaturation and low carotenoid content in the PVAT of the IMA of patients with more advanced coronary artery disease. Moreover, the spectral modelling of the IMA's PVAT composition indicated that glyceryl trioleate was a major PVAT lipid and for patients with relatively low levels of ß-carotene, it was accompanied by arachidonic acid and glyceryl trilinolenate. In summary, our proof-of-concept study suggests that carotenoid content and lipid unsaturation degree may reflect the PVAT functional status and a Raman-based assessment of the PVAT of the IMA could prove useful as a novel diagnostic tool to rapidly define the PVAT phenotype in a grafted artery in patients undergoing coronary bypass.

HPV Infection Significantly Accelerates Glycogen Metabolism in Cervical Cells with Large Nuclei: Raman Microscopic Study with Subcellular Resolution.

Using Raman microscopy, we investigated epithelial cervical cells collected from 96 women with squamous cell carcinoma (SCC) or belonging to groups I, IIa, IIID-1 and IIID-2 according to Munich III classification (IIID-1 and IIID-2 corresponding to Bethesda LSIL and HSIL groups, respectively). All women were tested for human papillomavirus (HPV) infection using PCR. Subcellular resolution of Raman microscopy enabled to understand phenotypic differences in a heterogeneous population of cervical cells in the following groups: I/HPV-, IIa/HPV-, IIa/HPV-, LSIL/HPV-, LSIL/HPV+, HSIL/HPV-, HSIL/HPV+ and cancer cells (SCC/HPV+). We showed for the first time that the glycogen content in the cytoplasm decreased with the nucleus size of cervical cells in all studied groups apart from the cancer group. For the subpopulation of large-nucleus cells HPV infection resulted in considerable glycogen depletion compared to HPV negative cells in IIa, LSIL (for both statistical significance, ca. 45%) and HSIL (trend, 37%) groups. We hypothesize that accelerated glycogenolysis in large-nucleus cells may be associated with the increased protein metabolism for HPV positive cells. Our work underlines unique capabilities of Raman microscopy in single cell studies and demonstrate potential of Raman-based methods in HPV diagnostics.

Distinct Chemical Changes in Abdominal but Not in Thoracic Aorta upon Atherosclerosis Studied Using Fiber Optic Raman Spectroscopy.

Fiber optic Raman spectroscopy and Raman microscopy were used to investigate alterations in the aorta wall and the surrounding perivascular adipose tissue (PVAT) in the murine model of atherosclerosis (Apoe-/-/Ldlr-/- mice). Both abdominal and thoracic parts of the aorta were studied to account for the heterogenic chemical composition of aorta and its localization-dependent response in progression of atherosclerosis. The average Raman spectra obtained for both parts of aorta cross sections revealed that the chemical composition of intima-media layers along aorta remains relatively homogeneous while the lipid content in the adventitia layer markedly increases with decreasing distance to PVAT. Moreover, our results demonstrate that the increase of the lipid to protein ratio in the aorta wall correlates directly with the increased unsaturation level of lipids in PVAT and these changes occur only in the abdominal, but not in thoracic, aorta. In summary, distinct pathophysiological response in the aortic vascular wall could be uncovered by fiber optic Raman spectroscopy based on simple parameters detecting chemical contents of lipids in PVAT.

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.

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 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.

Diversity among endothelial cell lines revealed by Raman and Fourier-transform infrared spectroscopic imaging.

Growing interest in the role of endothelium under physiological and pathological conditions has led to an increasing demand for its representative in vitro models especially suitable for drug tests and medical diagnostics. There are several endothelial cell lines commercially available whose biochemistry, and hence response to various stimuli, can be different. Recently, two vibrational techniques, Raman and Fourier-transform infrared microscopy, have been found to be potent tools for studying the biochemical composition of a single cell in an easy, rapid and label-free way. However, depending on the applied technique, the results may exhibit some divergence due to different selection rules as well as distinct experimental conditions. This paper presents the methodology of examination and characterization of three popular human endothelial cell lines: HAoEC (primary cells), HMEC-1 and EA.hy926 (immortalized cells). Based on high lateral resolution Raman imaging together with standard and high magnification Fourier-transform infrared measurements, the differences in spectral information and the distribution of biomolecules are presented and discussed.

Raman microscopy at the subcellular level: a study on early apoptosis in endothelial cells induced by Fas ligand and cycloheximide.

High spatially resolved Raman microscopy was applied to study the early apoptosis in endothelial cells and chemical and structural changes induced by this process. Application of cluster analysis enabled separation of signals due to various subcellular organelles and compartments such as the nuclei, nucleoli, endoplasmic reticulum or cytoplasm and analysis of alterations locally at the subcellular level. Different stimuli, i.e. Fas ligand, a tumor necrosis factor, and cycloheximide, an inhibitor of eukaryotic protein biosynthesis, were applied to induce apoptotic mechanisms. Due to different mechanisms of action, the changes observed in subcellular structures were different for FasL and cycloheximide. Although in both cases a statistically significant decrease of the protein level was observed in all studied cellular structures, the increase of the nucleic acids content locally in apoptotic nuclei was considerably more pronounced upon FasL-induced apoptosis compared to the cycloheximide one. Additionally, apoptosis invokes also a decrease of the proteins with the α-helix protein structure selectively for FasL in the cytoplasm and endoplasmic reticulum.

Raman microspectroscopy of human aortic valves: investigation of the local and global biochemical changes associated with calcification in aortic stenosis.

Raman microimaging was applied to study the biochemical composition in the aortic valves obtained from patients with calcific aortic stenosis. This progressive disease affects an increasing number of elderly patients with hyperlipidemia and hypercholesterolemia. Lipid accumulation in the tissue is associated with pathogenesis and progression of cardiac valve calcification. This is in line with our finding that lipid deposits, predominantly composed of cholesterol and its esters, are frequently co-localized with calcium salt deposits, even at an early stage of their development. Overall changes in the biochemical composition of the tissue upon pathology progression are less obvious. Globally, although the cholesterol level rises, the relative lipid-to-protein content decreases. The results broaden the knowledge of biochemical alterations in dysfunctional human aortic valves and may be helpful in designing lipid lowering therapies.

Imaging of perivascular adipose tissue in cardiometabolic diseases by Raman spectroscopy: Towards single-cell analysis.

Perivascular adipose tissue (PVAT) has emerged as a dynamic organ influencing vascular function and cardiovascular health. In this brief review, an overview of the recent research in the investigation of PVAT is presented, ranging from in vivo studies to single-cell methodologies, in particular those based on Raman spectroscopy. The strengths and limitations of each, emphasizing their contributions to the current understanding of PVAT biology were discussed. Ultimately, the integration of these diverse methodologies promises to uncover new therapeutic targets and diagnostic biomarkers, including those emerging from simple Raman spectroscopy-based measurements of alterations in lipid unsaturation degree, invariably associated with PVAT dysfunction.

Increased obesogenic action of palmitic acid during early stage of adipogenesis.

The functional differences between preadipocytes and fully differentiated mature adipocytes derived from stromal vascular fraction stem cells, as well as primary adipocytes have been analysed by evaluating their response to the obesogenic factor (a saturated fatty acid) and TNF-triggered inflammation. The analysis of single adipocytes shows that the saturated fatty acid (palmitic acid) accumulation is accompanied by inflammation and considerably dependent on the stage of the adipogenesis. In particular, preadipocytes show the exceptional potential for palmitic acid uptake resulting in their hypertrophy and the elevated cellular expression of the inflammation marker (ICAM-1). Our research provides new information on the impact of obesogenic factors on preadipocytes that is important in the light of childhood obesity prevention.

Direct oral and fiber-derived butyrate supplementation as an anti-obesity treatment via different targets.

BACKGROUND & AIMS: Butyric (one of the short-chain fatty acids), a major byproduct of the fermentation of non-digestible carbohydrates (e.g. fiber), is supposed to have anti-obesity and anti-inflammatory properties. However, butyrate's potential and mechanism in preventing obesity and the efficient form of administration remain to be clarified. METHODS: Hence, we studied the effect of oral supplementation with 5% (w/w) sodium butyrate and 4% (w/w) ß-glucan (fiber) on young male mice (C57BL/6J) with high-fat diet-induced obesity (HFD: 60 kcal% of fat + 1% of cholesterol). Six weeks old mice were fed diets based on HFD or control (AIN-93G) diet with/without supplements for 4 weeks. The unique, interdisciplinary approach combining several Raman-based techniques (including Raman microscopy and fiber optic Raman spectroscopy) and next-generation sequencing was used to ex vivo analyze various depots of the adipose tissue (white, brown, perivascular) and gut microbiome, respectively. RESULTS: The findings demonstrate that sodium butyrate more effectively prevent the pathological increase in body weight caused by elevated saturated fatty acids influx linked to a HFD in comparison to ß-glucan, thereby entirely inhibiting diet-induced obesity. Moreover, butyrate significantly affects the white adipose tissue (WAT) reducing the epididymal WAT mass in comparison to HFD without supplements, and decreasing lipid saturation in the epididymal WAT and perivascular adipose tissue of the thoracic aorta. Contrarily, ß-glucan significantly changes the composition and diversity of the gut microbiome, reversing the HFD effect, but shows no effect on the epididymal WAT mass and therefore the weight gain inhibition is not as effective as with sodium butyrate. CONCLUSIONS: Here, oral supplementation with sodium butyrate and ß-glucan (fiber) has been proven to have an anti-obesity effect through two different targets. Administration-dependent effects that butyrate imposes on the adipose tissue (oral administration) and microbiome (fiber-derived) make it a promising candidate for the personalized treatment of obesity.

Graphene Oxide Significantly Modifies Cardiac Parameters and Coronary Endothelial Reactivity in Healthy and Hypertensive Rat Hearts Ex Vivo.

Interactions of graphene oxide (GO) with an ex vivo rat heart and its coronary vessels have not been studied yet. Moreover, the conflicting data on the "structure-properties" relationships do not allow for biomedical applications of GO. Herein, we study the impact of GO on the ex vivo isolated rat heart, normotensive and hypertensive, under the working heart and the constant-pressure perfusion (Langendorff) regimes. Four structural GO variants of the following initial morphology were used: few-layer (below 10-layer) GO1, O < 49%; predominantly single-layer GO2, O = 41-50%; 15-20-layer GO3, O < 11%; and few-layer (below 10-layer) NH4 +-functionalized GO4, O < 44%, N = 3-6%. The aqueous GO dispersions, sonicated and stabilized with bovine serum albumin in Krebs-Henseleit-like solution-uniformized in terms of the particle size-were eventually size-monodisperse as revealed by dynamic light scattering. To study the cardiotoxicity mechanisms of GO, histopathology, Raman spectroscopy, analysis of cardiac parameters (coronary and aortic flows, heart rate, aortic pressure), and nitric oxide (NO-)-dependent coronary flow response to bradykinin (blood-vessel-vasodilator) were used. GO1 (10 mg/L) exerted no effects on cardiac function and preserved an increase in coronary flow in response to bradykinin. GO2 (10 mg/L) reduced coronary flow, aortic pressure in normotensive hearts, and coronary flow in hypertensive hearts, and intensified the response to bradykinin in normal hearts. GO3 (10 mg/L) reduced all parameters in hypertensive hearts and coronary response to bradykinin in normal hearts. At higher concentrations (normotensive hearts, 30 mg/L), the coronary response to bradykinin was blocked. GO4 (10 mg/L) reduced the coronary flow in normal hearts, while for hypertensive hearts, all parameters, except the coronary flow, were reduced and the coronary response to bradykinin was blocked. The results showed that a low number of GO layers and high O-content were safer for normal and hypertensive rat hearts. Hypertensive hearts deteriorated easier upon perfusion with low-O-content GOs. Our findings support the necessity of strict control over the GO structure during organ perfusion and indicate the urgent need for personalized medicine in biomedical applications of GO.

The impact of HPV infection on human glycogen and lipid metabolism - a review.

Reinterpretation of the Wartburg effect leads to understanding aerobic glycolysis as a process that provides considerable amount of molecular precursors for the production of lipids, nucleotides and amino acids that are necessary for continuous growth and rapid proliferation characteristic for cancer cells. Human papilloma virus (HPV) is a number one cause of cervical carcinoma with 99% of the cervical cancer patients being HPV positive. This tight link between HPV and cancer raises the question if and how HPV impact cells to reprogram their metabolism? Focusing on early phase proteins E1, E2, E5, E6 and E7 we demonstrate that HPV activates plethora of metabolic pathways and directly influences enzymes of the glycolysis pathway to promote the Warburg effect by increasing glucose uptake, activating glycolysis and pentose phosphate pathway, increasing the level of lactate dehydrogenase A synthesis and inhibiting ß-oxidation. Our considerations lead to conclusion that HPV is substantially involved in metabolic cell reprogramming toward neoplastic phenotype and its metabolic activity is the fundamental reason of its oncogenicity.

Raman studies of the adipose tissue: Current state-of-art and future perspectives in diagnostics.

The last decades revealed that the adipose tissue shows an unexplored therapeutic potential. In particular, targeting the perivascular adipose tissue (PVAT), that surrounds blood vessels, can prevent cardiovascular pathologies and browning of the adipose tissue can become an effective strategy against obesity. Therefore, new analytical tools are necessary to analyze this tissue. This review reports on the recent developments of various Raman-based techniques for the identification and quantification of the adipose tissue compared to conventional analytical methods. In particular, the emphasis is on analysis of PVAT, investigation of pathological changes of the adipose tissue in model systems and possibilities for its characterization in the clinical context. Overall, the review critically discusses the potential and limitations of Raman techniques in adipose tissue-targeted diagnostics and possible future anti-obesity therapies.