Agnieszka Jasztal

Visualization of the biochemical markers of atherosclerotic plaque with the use of Raman, IR and AFM.

In this work, we describe a methodology to visualize the biochemical markers of atherosclerotic plaque in cross sections of brachiocephalic arteries (BCA) taken from ApoE/LDLR(-/-) mice. The approach of the visualization of the same area of atherosclerotic plaque with the use of Raman, IR and AFM imaging enables the parallel characterisation of various features of atherosclerotic plaques. This support to the histochemical staining is utilized mainly in studies on mice models of atherosclerotic plaques, where micro and sub-micro resolutions are required. This work presents the methodology of the measurement and visualization of plaque features important for atherosclerosis development and plaques vulnerability analysis. Label-free imaging of cholesterol, cholesteryl esters, remodeled media, heme, internal elastic lamina, fibrous cap and calcification provides additional knowledge to previously presented quantitative measurements of average plaque features. AFM imaging enhanced the results obtained with the use of vibrational microspectroscopies with additional topographical information of the sample. To the best of our knowledge, this is the first work which demonstrates that co-localized measurement of atherosclerotic plaque with Raman, IR and AFM imaging provides a comprehensive insight into the biochemical markers of atherosclerotic plaques, and can be used as an integrated approach to assess vulnerability of the plaque.

The liver-selective NO donor, V-PYRRO/NO, protects against liver steatosis and improves postprandial glucose tolerance in mice fed high fat diet

BACKGROUND AND PURPOSE There is an unmet medical need for novel NAFLD treatments. Here we have examined the effects of liver-selective NO donor (V-PYRRO/NO) as compared with metformin on hepatic steatosis and glucose tolerance in mice fed high fat diet. MATERIAL AND METHODS Effects of V-PYRRO/NO (5 mgkg(-1)) or metformin (616 mgkg(-1)) were examined in C57BL/6J mice fed high fat diet (HF, 60 kcal% fat). Quantitative determination of steatosis, liver fatty acid composition and western blot analysis of selected proteins involved in mitochondrial biogenesis, fatty acid de novo synthesis and oxidation, triacylglycerols and cholesterol transport from the liver were performed. Liver NOx and nitrate concentration and blood biochemistry were also analyzed. RESULTS V-PYRRO/NO and metformin reduced liver steatosis with simultaneous reduction of total liver triacylglycerols, diacylglycerols and ceramides fraction and reversed HF-induced decrease in UFA/SFA ratio. V-PYRRO/NO substantially improved postprandial glucose tolerance, while the effect of metformin was modest and more pronounced on HOMA IR index. The anti-steatotic mechanism of V-PYRRO/NO was dependent on NO release, differed from that of metformin and involved improved glucose tolerance and inhibition of de novo fatty acid synthesis by Akt activation and ACC phosphorylation. In turn, major mechanism of metformin action involved increased expression of proteins implicated in mitochondrial biogenesis and metabolism (PGC-1α, PPARα, COX IV, cytochrome c, HADHSC). CONCLUSIONS V-PYRRO/NO acts as a liver-specific NO donor prodrug affording pronounced anti-steatotic effects and may represent an efficient, mechanistically novel approach to prevent liver steatosis and insulin resistance.

Antiatherosclerotic Effects of 1-Methylnicotinamide in Apolipoprotein E/Low-Density Lipoprotein Receptor–Deficient Mice: A Comparison with Nicotinic Acid

1-Methylnicotinamide (MNA), the major endogenous metabolite of nicotinic acid (NicA), may partially contribute to the vasoprotective properties of NicA. Here we compared the antiatherosclerotic effects of MNA and NicA in apolipoprotein E (ApoE)/low-density lipoprotein receptor (LDLR)–deficient mice. ApoE/LDLR−/− mice were treated with MNA or NicA (100 mg/kg). Plaque size, macrophages, and cholesterol content in the brachiocephalic artery, endothelial function in the aorta, systemic inflammation, platelet activation, as well as the concentration of MNA and its metabolites in plasma and urine were measured. MNA and NicA reduced atherosclerotic plaque area, plaque inflammation, and cholesterol content in the brachiocephalic artery. The antiatherosclerotic actions of MNA and NicA were associated with improved endothelial function, as evidenced by a higher concentration of 6-keto-prostaglandin F1α and nitrite/nitrate in the aortic ring effluent, inhibition of platelets (blunted thromboxane B2 generation), and inhibition of systemic inflammation (lower plasma concentration of serum amyloid P, haptoglobin). NicA treatment resulted in an approximately 2-fold higher concentration of MNA and its metabolites in urine and a 4-fold higher nicotinamide/MNA ratio in plasma, compared with MNA treatment. In summary; MNA displays pronounced antiatherosclerotic action in ApoE/LDLR−/− mice, an effect associated with an improvement in prostacyclin– and nitric oxide–dependent endothelial function, inhibition of platelet activation, inhibition of inflammatory burden in plaques, and diminished systemic inflammation. Despite substantially higher MNA availability after NicA treatment, compared with an equivalent dose of MNA, the antiatherosclerotic effect of NicA was not stronger. We suggest that detrimental effects of NicA or its metabolites other than MNA may limit beneficial effects of NicA-derived MNA.

Characterisation of atherogenic effects of low carbohydrate, high protein diet (LCHP) in apoE/LDLR−/− mice

IntroductionLow Carbohydrate High Protein diet represents a popular strategy to achieve weight loss.ObjectiveThe 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 MethodsAtherosclerosis 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.ResultsArea 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.ConclusionsThe 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.

Differential involvement of IL-6 in the early and late phase of 1-methylnicotinamide (MNA) release in Concanavalin A-induced hepatitis

Exogenous 1-methylnicotinamide (MNA) displays anti-inflammatory activity. The aim of this work was to characterize the profile of release of endogenous MNA during the initiation and progression of murine hepatitis induced by Concanavalin A (ConA). In particular we aimed to clarify the role of interleukin-6 (IL-6) as well as the energy state of hepatocytes in MNA release in early and late phases of ConA-induced hepatitis in mice. Hepatitis was induced by ConA in IL-6(+/+) and IL-6(-/-) mice, and various parameters of liver inflammation and injury, as well as the energy state of hepatocytes, were analysed in relation to MNA release. The decrease in ATP/ADP and NADH/NAD ratios, cytokine release (IL-6, IFN-ɤ), acute phase response (e.g. haptoglobin) and liver injury (alanine aminotransaminase, ALT) were all blunted in ConA-induced hepatitis in IL-6(-/-) mice as compared to IL-6(+/+) mice. The release of MNA in response to Con A was also significantly blunted in IL-6(-/-) mice as compared to IL-6(+/+) mice in the early stage of ConA-induced hepatitis. In turn, nicotinamide N-methyltransferase (NNMT) and aldehyde oxidase (AO) activities were blunted in the liver and MNA plasma concentration was elevated to similar degree in the late stage after Concanavalin A in IL-6(+/+) and IL-6(-/-) mice. In conclusion, we demonstrated that in ConA-induced hepatitis, early, but not late MNA release was IL-6-dependent. Our results suggest that in the initiation and early hepatitis, MNA release is linked to the energy deficit/impaired redox status in hepatocytes, while in a later phase, MNA release is rather linked to the systemic inflammation.

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.

Dual antiplatelet therapy with clopidogrel and aspirin increases mortality in 4T1 metastatic breast cancer-bearing mice by inducing vascular mimicry in primary tumour

Platelet inhibition has been considered an effective strategy for combating cancer metastasis and compromising disease malignancy although recent clinical data provided evidence that long-term platelet inhibition might increase incidence of cancer deaths in initially cancer-free patients. In the present study we demonstrated that dual anti-platelet therapy based on aspirin and clopidogrel (ASA+Cl), a routine regiment in cardiovascular patients, when given to cancer-bearing mice injected orthotopically with 4T1 breast cancer cells, promoted progression of the disease and reduced mice survival in association with induction of vascular mimicry (VM) in primary tumour. In contrast, treatment with ASA+Cl or platelet depletion did reduce pulmonary metastasis in mice, if 4T1 cells were injected intravenously. In conclusion, distinct platelet-dependent mechanisms inhibited by ASA+Cl treatment promoted cancer malignancy and VM in the presence of primary tumour and afforded protection against pulmonary metastasis in the absence of primary tumour. In view of our data, long-term inhibition of platelet function by dual anti-platelet therapy (ASA+Cl) might pose a hazard when applied to a patient with undiagnosed and untreated malignant cancer prone to undergo VM.

Vibrational Microspectroscopy for Analysis of Atherosclerotic Arteries

This Chapter describes the fundamental principles of atherosclerotic plaque formation along with features detectable by histology. The main focus is on reviewing the use of Raman and IR spectroscopy in the study of atherosclerotic arteries. The analysis of the experimental results is possible only with the application of mathematical and statistical tools of analysis, i.e. chemometrics, which are also described. This chapter proves that chemometrics allows us to create a link between IR and Raman spectroscopy and their application in chemistry.

Uptake and bioreactivity of charged chitosan-coated superparamagnetic nanoparticles as promising contrast agents for magnetic resonance imaging

Bioreactivity of superparamagnetic iron oxide nanoparticles (SPION) coated with thin layers of either cationic or anionic chitosan derivatives and serving as contrast agents in magnetic resonance imaging (MRI) was studied in vivo using BALB/c mouse model. Synthesized dual-modal fluorescing SPION were tracked in time using both fluorescent imaging and MRI. Although SPION started to be excreted by kidneys relatively shortly after administration they were uptaken by liver enhancing MRI contrast even up to 7 days. Importantly, chitosan-coated SPION caused only mild activation of acute phase response not affecting biochemical parameters of blood. Liver histology indicated the presence of SPION and modest increase in the number of Kupffer cells. The overall results indicated that SPION coated with ultrathin layers of chitosan ionic derivatives can serve as T2 contrast agents for diagnosis of liver diseases or imaging of other organs assuming the dose is optimized according to the need.

Anti-atherosclerotic effects of pravastatin in brachiocephalic artery in comparison with en face aorta and aortic roots in ApoE/LDLR−/− mice

BACKGROUND Cholesterol-dependent and independent mechanisms were proposed to explain anti-atherosclerotic action of statins in humans. However, their effects in murine models of atherosclerosis have not been consistently demonstrated. Here, we studied the effects of pravastatin on atherosclerosis in ApoE/LDLR-/- mice fed a control and atherogenic diet. METHODS ApoE/LDLR-/- mice were fed a control (CHOW) or an atherogenic (Low Carbohydrate High Protein, LCHP) diet. Two doses of pravastatin (40mg/kg and 100mg/kg) were used. The anti-atherosclerotic effects of pravastatin in en face aorta, cross-sections of aortic roots and brachiocephalic artery (BCA) were analysed. The lipid profile was determined. Fourier Transform Infrared Spectroscopy followed by Fuzzy C-Means (FCM) clustering was used for the quantitative assessment of plaque composition. RESULTS Treatment with pravastatin (100mg/kg) decreased total and LDL cholesterol only in the LCHP group, but displayed a pronounced anti-atherosclerotic effect in BCA and abdominal aorta. The anti-atherosclerotic effect of pravastatin (100mg/kg) in BCA was associated with significant alterations of the chemical plaque composition, including a fall in cholesterol and cholesterol esters contents independently on total cholesterol and LDL concentration in plasma. CONCLUSIONS Pravastatin at high (100mg/kg), but not low dose displayed a pronounced anti-atherosclerotic effect in ApoE/LDLR-/- mice fed a CHOW or LCHP diet that was remarkable in BCA, visible in en face aorta, whereas it was not observed in aortic roots, suggesting that previous inconsistencies might have been due to the various sites of atherosclerotic plaque analysis.