Vibrational spectroscopy-based quantification of liver steatosis.

The liver plays a central role in lipid metabolism, and abnormal lipid accumulation in the liver is a key feature of Non-Alcoholic Fatty Liver Disease. In experimental studies, quantification of liver steatosis is commonly based on lipids staining or biochemical analysis. Here, we present a spectroscopic approach for quantitative analysis of the lipid content in the freeze-dried liver. The method is based on vibrational spectroscopy (Raman and infrared) measurements applied for Partial Least Squares (PLS) regression modeling. The obtained PLS models show a good correlation of the spectroscopic data with the reference histological evaluation of steatosis based on Oil Red O (ORO)-stained images of liver cross sections. Vibrational spectroscopy with PLS-based modeling described here represents a useful approach for the fast assessment of the liver steatosis in a small sample of freeze-dried liver tissue. In conclusion, our work demonstrates the easy-to-use method that can be applied in laboratory routine as a beneficial alternative to the established ORO staining.

Alterations in NO- and PGI2- dependent function in aorta in the orthotopic murine model of metastatic 4T1 breast cancer: relationship with pulmonary endothelial dysfunction and systemic inflammation.

BACKGROUND: Patients with cancer develop endothelial dysfunction and subsequently display a higher risk of cardiovascular events. The aim of the present work was to examine changes in nitric oxide (NO)- and prostacyclin (PGI2)-dependent endothelial function in the systemic conduit artery (aorta), in relation to the formation of lung metastases and to local and systemic inflammation in a murine orthotopic model of metastatic breast cancer. METHODS: BALB/c female mice were orthotopically inoculated with 4T1 breast cancer cells. Development of lung metastases, lung inflammation, changes in blood count, systemic inflammatory response (e.g. SAA, SAP and IL-6), as well as changes in NO- and PGI2-dependent endothelial function in the aorta, were examined 2, 4, 5 and 6 weeks following cancer cell transplantation. RESULTS: As early as 2 weeks following transplantation of breast cancer cells, in the early metastatic stage, lungs displayed histopathological signs of inflammation, NO production was impaired and nitrosylhemoglobin concentration in plasma was decreased. After 4 to 6 weeks, along with metastatic development, progressive leukocytosis and systemic inflammation (as seen through increased SAA, SAP, haptoglobin and IL-6 plasma concentrations) were observed. Six weeks following cancer cell inoculation, but not earlier, endothelial dysfunction in aorta was detected; this involved a decrease in basal NO production and a decrease in NO-dependent vasodilatation, that was associated with a compensatory increase in cyclooxygenase-2 (COX-2)- derived PGI2 production. CONCLUSIONS: In 4 T1 metastatic breast cancer in mice early pulmonary metastasis was correlated with lung inflammation, with an early decrease in pulmonary as well as systemic NO availability. Late metastasis was associated with robust, cancer-related, systemic inflammation and impairment of NO-dependent endothelial function in the aorta that was associated with compensatory upregulation of the COX-2-derived PGI2 pathway.

Differential effects of nitric oxide deficiency on primary tumour growth, pulmonary metastasis and prostacyclin/thromboxane A2 balance in orthotopic and intravenous murine models of 4T1 breast cancer.

The role of nitric oxide (NO) in tumour progression and metastasis is not clear, therefore the present work aimed to better characterise the effects of nitric oxide synthase (NOS) inhibition by L-Nω-nitroarginine methyl ester (L-NAME) on primary tumour growth, pulmonary metastasis, inflammatory state and prostacyclin (PGI2)/thromboxane A2 (TXA2) balance in a 4T1 murine model of breast cancer. To distinguish effects of NO deficiency on disease development, 4T1 cancer cells were administered orthotopically or intravenously to Balb/c mice. The systemic NO bioavailability, pulmonary inflammation and plasma levels of thromboxane B2 (TXB2) and 6-keto-prostaglandin F1α (6-keto-PGF1α) were assessed. The study shows that, in the orthotopic model of 4T1 breast cancer, L-NAME hampered primary tumour growth, reduced pulmonary metastases, delayed inflammatory response but did not alter biosynthesis of TXB2 and 6-keto-PGF1α as well as PGI2/TXA2 ratio in cancer-bearing mice. Interestingly, in the intravenous model of 4T1 breast cancer, NOS inhibition did not influence metastasis nor inflammation, but it increased both TXB2 and 6-keto-PGF1α biosynthesis without affecting PGI2/TXA2 ratio. In conclusion, in a 4T1 murine model of metastatic breast cancer, NO plays a major role in primary tumour development, while NO is not the key mediator of cancer cell extravasation to the lungs. Furthermore, NO-deficiency activates a PGI2-dependent compensatory mechanism only in the intravenous model of 4T1 breast cancer.

Deletion of CD73 in mice leads to aortic valve dysfunction.

Aortic stenosis is known to involve inflammation and thrombosis. Changes in activity of extracellular enzyme - ecto-5'-nucleotidase (referred also as CD73) can alter inflammatory and thrombotic responses. This study aimed to evaluate the effect of CD73 deletion in mice on development of aortic valve dysfunction and to compare it to the effect of high-fat diet. Four groups of mice (normal-diet Wild Type (WT), high-fat diet WT, normal diet CD73-/-, high-fat diet CD73-/-) were maintained for 15weeks followed by echocardiographic analysis of aortic valve function, measurement of aortic surface activities of nucleotide catabolism enzymes as well as alkaline phosphatase activity, mineral composition and histology of aortic valve leaflets. CD73-/- knock out led to an increase in peak aortic flow (1.06±0.26m/s) compared to WT (0.79±0.26m/s) indicating obstruction. Highest values of peak aortic flow (1.26±0.31m/s) were observed in high-fat diet CD73-/- mice. Histological analysis showed morphological changes in CD73-/- including thickening and accumulation of dark deposits, proved to be melanin. Concentrations of Ca2+, Mg2+ and PO43- in valve leaflets were elevated in CD73-/- mice. Alkaline phosphatase (ALP) activity was enhanced after ATP treatment and reduced after adenosine treatment in aortas incubated in osteogenic medium. AMP hydrolysis in CD73-/- was below 10% of WT. Activity of ecto-adenosine deaminase (eADA), responsible for adenosine deamination, in the CD73-/- was 40% lower when compared to WT. Deletion of CD73 in mice leads to aortic valve dysfunction similar to that induced by high-fat diet suggesting important role of this surface protein in maintaining heart valve integrity.

Exercise capacity and cardiac hemodynamic response in female ApoE/LDLR(-/-) mice: a paradox of preserved V'O2max and exercise capacity despite coronary atherosclerosis.

We assessed exercise performance, coronary blood flow and cardiac reserve of female ApoE/LDLR(-/-) mice with advanced atherosclerosis compared with age-matched, wild-type C57BL6/J mice. Exercise capacity was assessed as whole body maximal oxygen consumption (V'O2max), maximum running velocity (vmax) and maximum distance (DISTmax) during treadmill exercise. Cardiac systolic and diastolic function in basal conditions and in response to dobutamine (mimicking exercise-induced cardiac stress) were assessed by Magnetic Resonance Imaging (MRI) in vivo. Function of coronary circulation was assessed in isolated perfused hearts. In female ApoE/LDLR(-/-) mice V'O2max, vmax and DISTmax were not impaired as compared with C57BL6/J mice. Cardiac function at rest and systolic and diastolic cardiac reserve were also preserved in female ApoE/LDLR(-/-) mice as evidenced by preserved fractional area change and similar fall in systolic and end diastolic area after dobutamine. Moreover, endothelium-dependent responses of coronary circulation induced by bradykinin (Bk) and acetylcholine (ACh) were preserved, while endothelium-independent responses induced by NO-donors were augmented in female ApoE/LDLR(-/-) mice. Basal COX-2-dependent production of 6-keto-PGF1α was increased. Concluding, we suggest that robust compensatory mechanisms in coronary circulation involving PGI2- and NO-pathways may efficiently counterbalance coronary atherosclerosis-induced impairment in V'O2max and exercise capacity.

Effects of Low Carbohydrate High Protein (LCHP) diet on atherosclerotic plaque phenotype in ApoE/LDLR-/- mice: FT-IR and Raman imaging.

Low Carbohydrate High Protein (LCHP) diet displays pro-atherogenic effects, however, the exact mechanisms involved are still unclear. Here, with the use of vibrational imaging, such as Fourier transform infrared (FT-IR) and Raman (RS) spectroscopies, we characterize biochemical content of plaques in Brachiocephalic Arteries (BCA) from ApoE/LDLR(-/-) mice fed LCHP diet as compared to control, recomended by American Institute of Nutrition, AIN diet. FT-IR images were taken from 6-10 sections of BCA from each mice and were complemented with RS measurements with higher spatial resolution of chosen areas of plaque sections. In aortic plaques from LCHP fed ApoE/LDLR(-/-) mice, the content of cholesterol and cholesterol esters was increased, while that of proteins was decreased as evidenced by global FT-IR analysis. High resolution imaging by RS identified necrotic core/foam cells, lipids (including cholesterol crystals), calcium mineralization and fibrous cap. The decreased relative thickness of the outer fibrous cap and the presence of buried caps were prominent features of the plaques in ApoE/LDLR(-/-) mice fed LCHP diet. In conclusion, FT-IR and Raman-based imaging provided a complementary insight into the biochemical composition of the plaque suggesting that LCHP diet increased plaque cholesterol and cholesterol esters contents of atherosclerotic plaque, supporting the cholesterol-driven pathogenesis of LCHP-induced atherogenesis.

Characterisation of Atherogenic Effects of Low Carbohydrate, High Protein Diet (LCHP) in ApoE/LDLR-/- Mice.

INTRODUCTION: Low Carbohydrate High Protein diet represents a popular strategy to achieve weight loss. OBJECTIVE: The aim of this study was to characterize effects of low carbohydrate, high protein diet (LCHP) on atherosclerotic plaque development in brachiocephalic artery (BCA) in apoE/LDLR-/- mice and to elucidate mechanisms of proatherogenic effects of LCHP diet. MATERIALS AND METHODS: Atherosclerosis plaques in brachiocephalic artery (BCA) as well as in aortic roots, lipoprotein profile, inflammation biomarkers, expression of SREBP-1 in the liver as well as mortality were analyzed in Control diet (AIN-93G) or LCHP (Low Carbohydrate High Protein) diet fed mice. RESULTS: Area of atherosclerotic plaques in aortic roots or BCA from LCHP diet fed mice was substantially increased as compared to mice fed control diet and was characterized by increased lipids and cholesterol contents (ORO staining, FT-IR analysis), increased macrophage infiltration (MOMA-2) and activity of MMPs (zymography). Pro-atherogenic phenotype of LCHP fed apoE/LDLR-/- mice was associated with increased plasma total cholesterol concentration, and in LDL and VLDL fractions, increased TG contents in VLDL, and a modest increase in plasma urea. LCHP diet increased SCD-1 index, activated SREBP-1 transcription factor in the liver and triggered acute phase response as evidence by an increased plasma concentration of haptoglobin, CRP or AGP. Finally, in long-term experiment survival of apoE/LDLR-/- mice fed LCHP diet was substantially reduced as compared to their counterparts fed control diet suggesting overall detrimental effects of LCHP diet on health. CONCLUSIONS: The pro-atherogenic effect of LCHP diet in apoE/LDLR-/- mice is associated with profound increase in LDL and VLDL cholesterol, VLDL triglicerides, liver SREBP-1 upregulation, and systemic inflammation.

Raman microimaging of murine lungs: insight into the vitamin A content.

The composition of the lung tissue of mice was investigated using Raman confocal microscopy at 532 nm excitation wavelength and was supported with various staining techniques as well as DFT calculations. This combination of experimental and theoretical techniques allows for the study of the distribution of lung lipofibroblasts (LIFs), rich in vitamin A, as well as the chemical structure of vitamin A. The comparison of the Raman spectra derived from LIFs with the experimental and theoretical spectra of standard retinoids showed the ability of LIFs to store all-trans retinol, which is partially oxidized to all-trans retinal and retinoic acid. Moreover, we were able to visualize the distribution of other lung tissue components including the surfactant and selected enzymes (lipoxygenase/glucose oxidase).