Chronic treatment with nitric oxide-releasing aspirin reduces plasma low-density lipoprotein oxidation and oxidative stress, arterial oxidation-specific epitopes, and atherogenesis in hypercholesterolemic mice

Abstract

The effects of chronic treatment with nitric oxide-containing aspirin (NO-aspirin, NCX-4016) in comparison with regular aspirin or placebo on the development of a chronic disease such as atherosclerosis were investigated in hypercholesterolemic low-density lipoprotein (LDL)-receptor-deficient mice. Male mice were assigned randomly to receive in a volume of 10 ml͞kg either placebo (n ‫؍‬ 10), 30 mg͞kg͞day NO-aspirin (n ‫؍‬ 10), or 18 mg͞kg͞day of regular aspirin (n ‫؍‬ 10). After 12 weeks of treatment, the computerassisted imaging analysis revealed that NO-aspirin reduced the aortic cumulative lesion area by 39.8 ؎ 12.3% compared with that of the placebo (P < 0.001). Regular aspirin did not reduce significantly aortic lesions (؊5.1 ؎ 2.3%) compared with the placebo [P ‫؍‬ 0.867, not significant (NS)]. Furthermore, NO-aspirin reduced significantly plasma LDL oxidation compared with aspirin and placebo, as shown by the significant reduction of malondialdehyde content (P < 0.001) as well as by the prolongation of lag-time (P < 0.01). Similarly, systemic oxidative stress, measured by plasma isoprostanes, was significantly reduced by treatment with NCX-4016 (P < 0.05). More importantly, mice treated with NO-aspirin revealed by immunohistochemical analysis of aortic serial sections a significant decrease in the intimal presence of oxidation-specific epitopes of oxLDL (E06 monoclonal antibody, P < 0.01), and macrophages-derived foam cells (F4͞80 monoclonal antibody, P < 0.05), compared with placebo or aspirin. These data indicate that enhanced NO release by chronic treatment with the NO-containing aspirin has antiatherosclerotic and antioxidant effects in the arterial wall of hypercholesterolemic mice.atherosclerosis ͉ LDL-receptor-deficient mice

Long-term combined beneficial effects of physical training and metabolic treatment on atherosclerosis in hypercholesterolemic mice

“…The experiments conformed to the National Institutes of Health guidelines for the care of laboratory animals and were carried out on six groups of 8-week-old male low-density lipoprotein (LDL) receptor-deficient hypercholesterolemic mice (mean weight 42.4 Ϯ 13.2 g). We selected only male mice to avoid gender-related differences (49,50). The mice were randomized to one of three dietary interventions: (i) High-cholesterol diet alone (containing 21% by weight fat, 0.15% by weight cholesterol, and 19.5% by weight casein; Harlan Teklad, Madison, WI), as described (49)(50)(51); (ii) high-cholesterol diet plus antioxidant supplementation (1.0% vitamin E added to the chow and 0.05% vitamin C added to the drinking water); and (iii) high-cholesterol diet plus both antioxidants and L-arginine (6% in drinking water).…”

“…Mice were killed by CO 2 asphyxia, and then the blood was drawn into heparinized tubes from the inferior vena cava. The dissection of the aorta was performed under a stereomicroscope, as described in detail (46,(49)(50)(51). A 2-mm cross section was saved for immunohistochemical analysis, as described (46,(49)(50)(51).…”

“…The dissection of the aorta was performed under a stereomicroscope, as described in detail (46,(49)(50)(51). A 2-mm cross section was saved for immunohistochemical analysis, as described (46,(49)(50)(51). Tissue sections (5 m) were incubated with the E06 antibody (a generous gift of W. Palinski and J. L. Witztum, University of California at San Diego, La Jolla) against oxidation specific epitopes (46, 50), or with F4͞80, an antibody against mouse macrophage-derived foam cells (46,50).…”

“…A 2-mm cross section was saved for immunohistochemical analysis, as described (46,(49)(50)(51). Tissue sections (5 m) were incubated with the E06 antibody (a generous gift of W. Palinski and J. L. Witztum, University of California at San Diego, La Jolla) against oxidation specific epitopes (46, 50), or with F4͞80, an antibody against mouse macrophage-derived foam cells (46,50). The aorta was then cut longitudinally; one half was placed in 250 l of protein extraction buffer, and the other half was saved in 1 ml of KH 2 PO 4 buffer (46, 49-51) for further determination of eNOS protein expression (Western blot with rabbit polyclonal anti-human eNOS antibody) and the signal was detected by using chemiluminescence (Amersham Pharmacia ECL) as described (46,(49)(50)(51).…”

“…Cholesterolemia was determined enzymatically (50). LDL particles (d ϭ 1.006-1.063 g͞ml) were isolated from pooled plasma from two animals from each group by sequential-density ultracentrifugation, as described (50,53).…”