Lukasz Mateuszuk

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.

Mildronate, a Regulator of Energy Metabolism, Reduces Atherosclerosis in apoE/LDLR–/– Mice

Background/Aims: Mildronate, an inhibitor of L-carnitine biosynthesis and transport, is used in clinics as a modulator of cellular energy metabolism and is a cardioprotective drug. L-Carnitine is a pivotal molecule in fatty acid oxidation pathways and its regulation in vasculature might be a promising approach for antiatherosclerotic treatment. This study was performed to evaluate the effects of mildronate treatment on the progression of atherosclerosis and the content of L-carnitine in the vascular wall. Methods: ApoE/LDLR–/– mice received mildronate at doses of 30 and 100 mg/kg for 4 months. Lipid profile was measured in plasma and atherosclerotic lesions were analyzed in whole aorta and aortic sinus. L-Carnitine concentration was assessed in rat aortic tissues after 2 weeks of treatment with mildronate at a dose of 100 mg/kg. Results: The chronic treatment with mildronate at a dose of 100 mg/kg significantly reduced the size of atherosclerotic plaques in the aortic roots and in the whole aorta, and slightly decreased the free cholesterol level. In addition, mildronate treatment decreased L-carnitine concentration in rat aortic tissues. Conclusions: Long-term mildronate treatment decreases L-carnitine content in aortic tissues and attenuates the development of atherosclerosis in apoE/LDLR–/– mice.

Functional alterations in endothelial NO, PGI2 and EDHF pathways in aorta in ApoE/LDLR−/− mice

Adequate endothelial production of nitric oxide (NO), endothelium-derived hyperpolarizing factor (EDHF), and prostacyclin (PGI₂) is critical to the maintenance of vascular homeostasis. However, it is not clear whether alterations in each of these vasodilatory pathways contribute to the impaired endothelial function in murine atherosclerosis. In the present study, we analyze the alterations in NO-, EDHF- and PGI₂-dependent endothelial function in the thoracic aorta in relation to the development of atherosclerotic plaques in apoE/LDLR⁻/⁻ mice. We found that in the aorta of 2-month-old apoE/LDLR⁻/⁻ mice there was no lipid deposition, subendothelial macrophage accumulation; and matrix metalloproteinase (MMP) activity was low, consistent with the absence of atherosclerotic plaques. Interestingly, at this stage the endothelium was already activated and hypertrophic as evidenced by electron microscopy, while acetylcholine-induced NO-dependent relaxation in the thoracic aorta was impaired, with concomitant upregulation of cyclooxygenase-2 (COX-2)/PGI₂ and EDHF (epoxyeicosatrienoic acids, EETs) pathways. In the aorta of 3-6-month-old apoE/LDLR⁻/⁻ mice, lipid deposition, macrophage accumulation and MMP activity in the intima were gradually increased, while impairment of NO-dependent function and compensatory upregulation of COX-2/PGI₂ and EDHF pathways were more accentuated. These results suggest that impairment of NO-dependent relaxation precedes the development of atherosclerosis in the aorta and early upregulation of COX-2/PGI₂ and EDHF pathways may compensate for the loss of the biological activity of NO.

Endothelium in Spots – High-Content Imaging of Lipid Rafts Clusters in db/db Mice

Lipid rafts (LRs) are dynamic, sterol- and sphingolipid-enriched nanodomains involved in the regulation of cellular functions and signal transduction, that upon stimuli, via (e.g. association of raft proteins and lipids), may cluster into domains of submicron or micron scale. Up to date, however, lipid raft clusters were observed only under artificially promoted conditions and their formation in vivo has not been confirmed. Using non-destructive approach involving Raman and Atomic Force Microscopy imaging we demonstrated the presence of clustered lipid rafts in endothelium of the aorta of the db/db mice that represent a reliable murine model of type 2 diabetes. The raft clusters in the aorta of diabetic mice were shown to occupy a considerably larger (about 10-fold) area of endothelial cells surface as compared to the control. Observation of pathology-promoted LRs confirms that the cellular increase of lipid content results in clustering of LRs. Clustering of LRs leads to the formation of assemblies with diameters up to 3 micrometers and increased lipid character. This massive clustering of lipid rafts in diabetes may trigger a signaling cascade leading to vascular inflammation.

Quantification of plaque area and characterization of plaque biochemical composition with atherosclerosis progression in ApoE/LDLR−/− mice by FT-IR imaging

In this work the quantitative determination of atherosclerotic lesion area (ApoE/LDLR(-/-) mice) by FT-IR imaging is presented and validated by comparison with atherosclerotic lesion area determination by classic Oil Red O staining. Cluster analysis of FT-IR-based measurements in the 2800-3025 cm(-1) range allowed for quantitative analysis of the atherosclerosis plaque area, the results of which were highly correlated with those of Oil Red O histological staining (R(2) = 0.935). Moreover, a specific class obtained from a second cluster analysis of the aortic cross-section samples at different stages of disease progression (3, 4 and 6 months old) seemed to represent the macrophages (CD68) area within the atherosclerotic plaque.

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.

Simultaneous quantification of PGI2 and TXA2 metabolites in plasma and urine in NO-deficient mice by a novel UHPLC/MS/MS method.

The balance between vascular prostacyclin (PGI2) generated mainly via cyclooxygenase-2 (COX-2) and its physiological antagonist platelet-derived thromboxane A2 (TXA2) formed by cyclooxygenase-1 (COX-1) determines cardiovascular homeostasis. In the present work, a novel bioanalytical method for simultaneous quantification of stable plasma and urinary metabolites of PGI2 (6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α) and TXA2 (TXB2, 2,3-dinor-TXB2) using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was developed. The method was validated using artificial plasma and urine and linearity range, intra- and inter-day precision and accuracy, recovery of analytes, relative and absolute matrix effect and stability of analytes were determined. The use of artificial biofluids improved the method sensitivity as it eliminated the contribution of endogenous metabolites present in mice plasma and urine to validation procedure. The newly developed and validated method allowed to quantify 6-keto-PGF1α and TXB2 in mice plasma as well as 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 in urine samples with high sensitivity and accuracy. The calibration range was established from 0.1 to 100ng/mL for all analytes using artificial biofluids and the recoveries were greater than 89.9%. All validated parameters met the criteria of acceptance specified in FDA and EMA guidance. This method was successfully employed for profiling of the changes in PGI2 and TXA2 generation in NO-deficient mice. This work demonstrated that NO-deficiency induced by L-NAME, evidenced by a fall in nitrite in plasma and urine, was associated with platelet activation, robust increase in TXB2 and mild increase in 6-keto-PGF1α concentration in plasma. Changes in 2,3-dinor-6-keto-PGF1α and 2,3-dinor-TXB2 concentration in urine were less evident suggesting that the measurements in plasma better reflect modest changes in PGI2/TXA2 homeostasis than measurements in urine.

Multi-methodological insight into the vessel wall cross-section: Raman and AFM imaging combined with immunohistochemical staining

The vascular wall cross-section was submitted to multiparametric analysis with the application of several micro-scale methods: Raman and atomic force microscopy (AFM) imaging as well as immunohistochemical (IHC) staining. Both IHC staining as a standard reference method and Raman imaging allowed for visualization of tissue components such as elastin fibers or cell nuclei, moreover, Raman spectroscopy provided specific biochemical information about sample composition. Application of AFM complemented the chemical information with the data about the topography of the studied sample. Developing such a methodology allows for the multiparameter description of the vessel wall status that could be useful to monitor ex vivo the disease progression and effects of treatment in experimental diabetes, atherosclerosis or hypertension.

Triple immunofluorescence labeling of atherosclerotic plaque components in apoE/LDLR -/- mice

This paper presents a simple and reliable method of triple immunofluorescence staining that allows simultaneous detection of various cell types present in atherosclerotic plaque of apolipoprotein E and LDL receptor-double knockout (apoE/LDLR -/-) mice. We used combined direct and indirect procedures applying commercially available primary antibodies raised in different species to detect smooth muscle cells (Cy3-conjugated mouse anti-smooth muscle actin, SMA), macrophages (rat anti-CD68) and T lymphocytes (rabbit anti-CD3). Fixation of the material in acetone and modified incubation protocol employing nonfat dry milk in preincubation and incubation media significantly increased the intensity of labeling and effectively quenched the background. Our method offers an efficient way to detect qualitative as well as quantitative changes of macrophages, T lymphocytes and smooth muscle cells in atherosclerotic plaque of apoE/LDLR -/- mice during atherosclerosis development or in response to pharmacological treatment.

Alterations in plasma biochemical composition in NO deficiency induced by L‐NAME in mice analysed by Fourier Transform Infrared Spectroscopy

Mouse model of nitric oxide deficiency, induced by prolonged treatment with NG -nitro-L-arginine methyl ester (L-NAME) was used for infrared spectroscopy (FTIR) analysis of plasma. L-NAME leads to increased peripheral resistance and systemic hypertension. Classification of spectral response was by principal component analysis (PCA) and linear discriminant analysis (LDA). PCA allowed to separate each animal group showing that FTIR spectra are sensitive to development of NO-deficiency on contrary to blood pressure values indicating hypertension. Globally, the most pronounced spectral alternations were observed in the second and third week of L-NAME treatment indicating that infrared signature of blood plasma can serve as indicator of early and late stages of the disease. The PLS-DA method provided >95% classification accuracy. Spectral features characteristic for L-NAME treatment were mainly associated with an elevated level of proteins accompanied by a decrease of a tyrosine content and changes in lipids/phospholipid concentration. In our work we discuss these changes for which statistically significant differences (p < 0.05 - 0.005) were observed between spectra collected for each time-point of the L-NAME treatment versus control subjects. We demonstrated for the first time that NO-deficiency and hypertension resulted in changes in biochemical profile of plasma that was detected by FTIR spectroscopy.