Fixed versus live endothelial cells: The effect of glutaraldehyde fixation manifested by characteristic bands on the Raman spectra of cells.

This work shows an impact of glutaraldehyde (GA) fixation on endothelial cells. Raman spectroscopy imaging was used as a method to monitor biochemical content of the cells due to GA fixation since this is an approach frequently used for studying cells by means of Raman imaging. To get a deeper insight into the changes and to understand them better the measurements of live and fixed cells were performed using two lasers, i.e. 488 and 532 nm. It has been demonstrated that GA fixation affects lipids, proteins, nucleic acid and carbohydrates to small extent. The application of 488 nm laser line seems to be more efficient for live cells due to the small impact of cytochrome resonance on Raman spectra, however 532 nm line is more beneficial for fixed cells due to higher quantum efficiency of the detector, thus leading to higher intensity of Raman bands. Generally, the changes due to fixation are not pronounced but cannot be ignored and the knowledge about them can help in a proper interpretation of data collected for fixed versus live cells.

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.

Effects of chronic nitric oxide synthase inhibition on V'O2max and exercise capacity in mice.

Acute inhibition of NOS by L-NAME (Nω-nitro-L-arginine methyl ester) is known to decrease maximal oxygen consumption (V'O2max) and impair maximal exercise capacity, whereas the effects of chronic L-NAME treatment on V'O2max and exercise performance have not been studied so far. In this study, we analysed the effect of L-NAME treatment, (LN2 and LN12, respectively) on V'O2max and exercise capacity (in maximal incremental running and prolonged sub-maximal incremental running tests), systemic NO bioavailability (plasma nitrite (NO2-) and nitrate (NO3-)) and prostacyclin (PGI2) production in C57BL6/J mice. Mice treated with L-NAME for 2 weeks (LN2) displayed higher V'O2max and better running capacity than age-matched control mice. In LN2 mice, NO bioavailability was preserved, as evidenced by maintained NO2- plasma concentration. PGI2 production was activated (increased 6-keto-PGF1α plasma concentration) and the number of circulating erythrocytes (RBC) and haemoglobin concentration were increased. In mice treated with L-NAME for 12 weeks (LN12), NO bioavailability was decreased (lower NO2- plasma concentration), and 6-keto-PGF1α plasma concentration and RBC number were not elevated compared to age-matched control mice. However, LN12 mice still performed better during the maximal incremental running test despite having lower V'O2max. Interestingly, the LN12 mice showed poorer running capacity during the prolonged sub-maximal incremental running test. To conclude, short-term (2 weeks) but not long-term (12 weeks) treatment with L-NAME activated robust compensatory mechanisms involving preservation of NO2- plasma concentration, overproduction of PGI2 and increased number of RBCs, which might explain the fully preserved exercise capacity despite the inhibition of NOS.

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.

Identification of a biochemical marker for endothelial dysfunction using Raman spectroscopy.

In the present work, we propose the spectroscopic approach to identify biochemical alterations in endothelial dysfunction. The method is based on the quantification of the ratio of phenylalanine (Phe) to tyrosine (Tyr) contents in the endothelium. The synthesis of Tyr from Phe requires the presence of tetrahydrobiopterin (BH4) as a cofactor of phenylalanine hydroxylase (PAH). Limitation of BH4 availability in the endothelium is a hallmark endothelial nitric oxide synthase (eNOS) dysfunction that may also lead to PAH dysfunction and a fall in Tyr contents. Using Raman spectra, the ratio of marker bands of Tyr to Phe was calculated and the pathological state of the endothelium was detected. We provide evidence that Phe/Tyr ratio analysis by Raman spectroscopy discriminate endothelial dysfunction in ApoE/LDLR(-/-) mice as compared to control mice.

Biochemical changes of the endothelium in the murine model of NO-deficient hypertension.

The main spectral differences between the biochemical compositions of the vascular endothelium of control, hypertensive NO-deficient, and NO-deficient mice supplemented with nitrate were studied using Raman microimaging. A significantly different Raman signature of the endothelium in these three groups in the 1200-1400 cm(-1) region was assigned to the α-helix and ß-sheet alterations in the protein secondary structure upon the development of hypertension. The second pronounced biochemical marker of endothelium alterations was the lipid to protein ratio. A lower intensity of the band at 2940 cm(-1) relative to the feature at 1007 cm(-1) in the endothelium in hypertension compared to the control indicated a decrease of the lipid content relative to proteins during the progress of the pathology. The nitrate-based treatment partially reversed the effects of hypertension. The nitrate supplementation restored the lipid to protein ratio in the endothelium to the control level, while the changes in the secondary structure of proteins were irreversible upon nitrate administration.

Effects of margarine supplemented with t10c12 and C9T11 CLA on atherosclerosis and steatosis in apoE/LDLR -/- mice.

OBJECTIVES: The objective of this study was to evaluate functional effects of margarine supplemented with individual CLA isomers trans-10, cis-12 and cis-9, trans-11 in apoE/LDLR -/- mice. DESIGN: In LONG experiment (LONG), two-month old mice with no atherosclerosis were assigned to experimental groups and fed for the next 4 months. In SHORT experiment (SHORT), four-month old mice, with pre-established atherosclerosis, were assigned to experimental groups and fed for the next 2 months. The experimental diets were: AIN-93G (margarine), AIN-93G + 0.5% trans-10, cis-12 CLA (t10c12), and AIN-93G + 0.5% cis-9, trans-11 CLA (c9t11). RESULTS: In both experiments (LONG and SHORT), liver weight was significantly (P<0.05) increased in mice fed t10c12 CLA. Hepatic steatosis was found in animals fed t10c12 diet and no signs of the steatosis was observed in mice fed c9t11 CLA. Dietary treatments with t10c12 CLA significantly increased total plasma cholesterol and plasma triacylglycerols. There were no isomer-specific effects of CLA isomers on area of atherosclerotic plaque in aortic root. CONCLUSION: In conclusion, t10c12 CLA significantly increased liver weight in mice in LONG and SHORT experiments. Our results do not support the notion that CLA isomer supplementation to the margarine possess anti-atheroclerotic effect. Therefore, no isomer-specific effects of CLA on development of atherosclerosis were observed.

Inhibition of five lipoxygenase activating protein (FLAP) by MK-886 decreases atherosclerosis in apoE/LDLR-double knockout mice.

BACKGROUND: Recent reports point to an important role of leukotrienes in atherogenesis. Leukotrienes are produced by 5-lipoxygenase co-operating with five lipoxygenase activating protein (FLAP). We hypothesized that MK-886, an inhibitor of FLAP, could attenuate the development of atherosclerosis in the atherogenic apolipoprotein E/low density lipoprotein receptor (apoE/LDLR) double knockout (DKO) mouse model. MATERIALS AND METHODS: Female apoE/LDLR-DKO mice at the age of 8 weeks were put on Western diet. The experimental group (n = 10) received the same diet as the control group (n = 10), but mixed with MK-886 (Merck, Rahway, NJ) at a dose of 4 microg per 100 mg of body-weight per day. At age 6 months the mice were sacrificed under anaesthesia. RESULTS: Measured by the en face method, the percentage of area occupied by lesions in aortas in the control group was 25.15 +/- 2.9%, whereas in the MK-886-treated group it was 11.16 +/- 0.7% (P < 0.05). Lesion area measured by cross-section of aortic roots was 455,494 +/- 29,564 microm(2) in the control group versus 263,042 +/- 20,736 microm(2) in the MK-886-treated group (P < 0.05). The MK-886 did not change the plasma cholesterol lipoprotein profile as compared with the control mice. Finally, we show that MK-886 may increase plaque stability by decreasing the macrophage content as well as increasing the collagen and smooth-muscle cell content. CONCLUSIONS: Our results show for the first time that inhibition of FLAP by MK-886 reduces development of atherosclerosis in gene-targeted apoE/LDLR-DKO mice.

Inhibition of nuclear factor-kappaB attenuates artherosclerosis in apoE/LDLR - double knockout mice.

Nuclear factor - kappaB (NF-kappaB) is a good therapeutic target for cardiovascular disease and numerous efforts are being made to develop safe NF-kappaB inhibitors. Nowadays many authors address NF-kappaB as a major therapeutic target in atherosclerosis, especially for preventive measures, in the light of two main hypothesis of atherosclerosis: oxidation and inflammation. We hypothesized that ammonium pyrrolidinedithioocarbamate (PDTC) - a well-known inhibitor of NF-kappaB could inhibit the development of atherosclerosis in this experimental model. We used apoE/LDLR - DKO mouse model, which is considered as a one of the best models to study the anti-atherosclerotic effect of drugs. In this model PDTC inhibited atherogenesis, measured both by "en face" method (25,15+/-2,9% vs. 15,63+/-0,6%) and "cross-section" method (565867+/-39764 microm2 vs. 291695+/-30384 microm2). Moreover, PDTC did not change the profile of cholesterol and triglycerides in blood. To our knowledge, this is the first report that shows the effect of PDTC on atherogenesis in gene-targeted apoE/LDLR - double knockout mice.

Effect of curcumin on atherosclerosis in apoE/LDLR-double knockout mice.

It is widely appreciated that inflammation and oxidant stress contribute to atherogenesis. Curcumin, a polyphenolic natural compound has been reported to possess anti-inflammatory and anti-oxidant actions. We hypothesized that curcumin could inhibit the development of atherosclerosis in the apoE/LDLR-double knockout mice fed with Western diet (21% fat, 0.15% cholesterol w/w, without cholic acid). Curcumin (purity>or=98%), premixed with diet, was given for 4 months at a dose of 0.3 mg/ per day/ per mouse. In this model curcumin inhibited atherogenesis, measured both by "en face" method (25,15+/-2,9% vs. 19,2+/-0,6%, p<0,05) and "cross-section" method (565867+/-39764 microm2 vs. 299201+/-20373 microm2, p<0,05). Importantly, curcumin influenced neither the concentrations of cholesterol and triglycerides in blood nor animal body weight. To our knowledge, this is the first report that shows the anti-atherogenic effect of low dose of curcumin in fine model of atherosclerosis: gene-targeted apoE/LDLR-double knockout mice.