Dietary carnosine prevents early atherosclerotic lesion formation in apolipoprotein E-null mice - PubMed (original) (raw)

Comparative Study

Dietary carnosine prevents early atherosclerotic lesion formation in apolipoprotein E-null mice

Oleg A Barski et al. Arterioscler Thromb Vasc Biol. 2013 Jun.

Abstract

Objective: Atherosclerotic lesions are associated with the accumulation of reactive aldehydes derived from oxidized lipids. Although inhibition of aldehyde metabolism has been shown to exacerbate atherosclerosis and enhance the accumulation of aldehyde-modified proteins in atherosclerotic plaques, no therapeutic interventions have been devised to prevent aldehyde accumulation in atherosclerotic lesions.

Approach and results: We examined the efficacy of carnosine, a naturally occurring β-alanyl-histidine dipeptide, in preventing aldehyde toxicity and atherogenesis in apolipoprotein E-null mice. In vitro, carnosine reacted rapidly with lipid peroxidation-derived unsaturated aldehydes. Gas chromatography mass-spectrometry analysis showed that carnosine inhibits the formation of free aldehydes 4-hydroxynonenal and malonaldialdehyde in Cu(2+)-oxidized low-density lipoprotein. Preloading bone marrow-derived macrophages with cell-permeable carnosine analogs reduced 4-hydroxynonenal-induced apoptosis. Oral supplementation with octyl-D-carnosine decreased atherosclerotic lesion formation in aortic valves of apolipoprotein E-null mice and attenuated the accumulation of protein-acrolein, protein-4-hydroxyhexenal, and protein-4-hydroxynonenal adducts in atherosclerotic lesions, whereas urinary excretion of aldehydes as carnosine conjugates was increased.

Conclusions: The results of this study suggest that carnosine inhibits atherogenesis by facilitating aldehyde removal from atherosclerotic lesions. Endogenous levels of carnosine may be important determinants of atherosclerotic lesion formation, and treatment with carnosine or related peptides could be a useful therapy for the prevention or the treatment of atherosclerosis.

Keywords: aldehydes; atherosclerosis; carnosine; oxidized low-density lipoprotein.

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Figures

Figure 1

Figure 1. Histidyl dipeptides prevent LDL oxidation

Oxidation of LDL (measured by the formation of TBARS) in the presence of A, CuSO4 (10 μM) and 10 mM carnosine (CAR), anserine (ANS), or homocarnosine (HOMO) or B, murine bone marrow-derived macrophages (BMM), and carnosine or N-acetyl-carnoine (NAcCAR). For the experiments with N-acetyl-carnosine, BMM were pre-incubated with NacCAR (10 mM) for 24 h and rinsed with PBS prior to the addition of LDL. C, Formation of TBARS (i), hydroperoxides (ii), and conjugated dienes (iii) in LDL oxidized in the presence of Cu2+ and 1 or 10 mM carnosine. D. Inhibition of HNE and MDA formation in Cu2+-oxidized LDL in the presence of carnosine. Inset shows GC-CI/MS identification of MDA and HNE. Values are mean ± SE from 3 independent experiments. *P<0.05.

Figure 2

Figure 2. Carnosine reacts with oxidized lipids-derived aldehydes

The rate of disappearance of A, HNE and B, HHE (10 μM each) from a reaction mixture containing indicated concentrations of carnosine in 0.15 M potassium phosphate, pH 7.4. The reaction mixture was incubated at 37°C and aliquots were removed at indicated times to measure free aldehyde by GC-CI/MS. Data are shown as discrete points and the curves are the best fit of a single exponential equation to the data [_y=Ae−kobst_]. C, Concentration dependence of kobs. Best fits of the linear dependence were used to calculate the second order rate constants listed in Table 1. ACR indicates acrolein; OCT, octenal; ONE, 4-oxononenal.

Figure 3

Figure 3. Carnosine protects macrophages from HNE-induced apoptosis

Flow cytometry analysis of bone marrow macrophages treated with A, HNE or carnosine-HNE or B, pre-incubated with the cell-permeable carnosine analogs, octyl-D-carnosine (ODC) or N-acetyl-carnosine (NacCAR) for 16h and then incubated for 2h with HBSS or HNE as indicated. Cells were labeled with FITC-Annexin V/7-AAD to monitor apoptosis. The lower right quadrant shows the early apoptotic cells and the upper right quadrant shows the late apoptotic/necrotic cells. Panel C shows the effect of ODC and NacCAR on HNE-induced apoptosis in the presence of 5% fetal bovine serum in RPMI medium. Values are mean ± SEM of 3 independent experiments, each conducted in triplicate or quadruplicate. *P<0.01 versus HNE-treated cells.

Figure 4

Figure 4. Carnosine-feeding inhibits early phase of atherogenesis and decreases the accumulation of macrophages in lesions

A, Carnosine level in the plasma (i), spleen (ii) and aorta (iii) of 14-week-old female apoE-null mice maintained on high fat diet and fed ODC (60 mg/kg in drinking water) for 6 weeks starting at 8 weeks of age. Concentration of carnosine was measured by LC/ESI+-MS as described in Methods. B, Lesions in the aortic valve. Lipids were visualized by Oil red O staining. Panels (i) shows the representative photomicrographs of aortic valve of control and ODC-fed mice. Panel (ii) shows the group data. C, Macrophage accumulation in the aortic valve was examined by staining with Alexa 647-conjugated CD-68 antibody. Panels (i) and (ii) shows the representative photomicrographs of aortic valve of control and ODC-fed mice and the group data, respectively. Values are means ± SEM. *P < 0.05 versus controls.

Figure 5

Figure 5. Carnosine removes aldehydes from atherosclerotic lesions of apoE-null mice

Photomicrographs of aortic valves stained with A, anti-KLH-acrolein B, anti-KLH-HHE, or C, anti-KLH-HNE antibodies. Mice were treated with ODC as described in Fig. 4. Nuclei were stained with DAPI. Values are means ± SEM. #P < 0.01 versus controls. D, Abundance of CAR-HNE and CAR-HHE conjugates in the urine of apoE-null mice, described in panels A-C. Urine was collected 2 days prior to sacrifice. E, Abundance of carnosine-HHE in the urine of 30 week-old female C57BL/6 and apoE-null mice before and after feeding ODC. Control urine was collected for 24 hours. Mice were then fed ODC (60 mg/kg in water by gavage) and urine was collected for an additional 24 hours. MRM traces of the conjugates are shown in Supplemental Figure IV. Values are means ± SEM. *P < 0.05.

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