Endothelial dysfunction in a murine model of mild hyperhomocyst(e)inemia

Abstract

Homocysteine is a risk factor for the development of atherosclerosis and its thrombotic complications. We have employed an animal model to explore the hypothesis that an increase in reactive oxygen species and a subsequent loss of nitric oxide bioactivity contribute to endothelial dysfunction in mild hyperhomocysteinemia. We examined endothelial function and in vivo oxidant burden in mice heterozygous for a deletion in the cystathionine beta-synthase (CBS) gene, by studying isolated, precontracted aortic rings and mesenteric arterioles in situ. CBS(-/+) mice demonstrated impaired acetylcholine-induced aortic relaxation and a paradoxical vasoconstriction of mesenteric microvessels in response to superfusion of methacholine and bradykinin. Cyclic GMP accumulation following acetylcholine treatment was also impaired in isolated aortic segments from CBS(-/+) mice, but aortic relaxation and mesenteric arteriolar dilation in response to sodium nitroprusside were similar to wild-type. Plasma levels of 8-epi-PGF(2alpha) (8-IP) were somewhat increased in CBS(-/+) mice, but liver levels of 8-IP and phospholipid hydroperoxides, another marker of oxidative stress, were normal. Aortic tissue from CBS(-/+) mice also demonstrated greater superoxide production and greater immunostaining for 3-nitrotyrosine, particularly on the endothelial surface. Importantly, endothelial dysfunction appears early in CBS(-/+) mice in the absence of structural arterial abnormalities. Hence, mild hyperhomocysteinemia due to reduced CBS expression impairs endothelium-dependent vasodilation, likely due to impaired nitric oxide bioactivity, and increased oxidative stress apparently contributes to inactivating nitric oxide in chronic, mild hyperhomocysteinemia.

Figure 1

(a) Total plasma homocysteine concentration in CBS^+/+ mice (n = 5) and CBS^–/+ mice (n = 7), and (b) CBS activity from liver homogenates in CBS^+/+ mice (n = 6) and CBS^–/+ mice (n = 6). ^AP < 0.05.

Figure 2

Relaxation of isolated, precontracted aortic rings in response to acetylcholine (a) and sodium nitroprusside (b) in CBS^+/+ mice (circles; n = 9) and CBS^–/+ mice (squares; n = 9). ^AP < 0.05; ^BP < 0.001; ^CP = NS.

Figure 3

Mesenteric microvascular response to superfusion of methacholine (a), bradykinin (b), and sodium nitroprusside (c) in CBS^+/+ mice (circles) (n = 4) and CBS^–/+ mice (squares) (n = 4). ^AP < 0.05; ^BP < 0.001; ^CP = NS.

Figure 4

Aortic cyclic GMP accumulation stimulated with acetylcholine for 1 minute in CBS^+/+ mice (n = 4) and CBS^–/+ mice (n = 4). ^AP = 0.15.

Figure 5

Plasma 8-isoprostane levels from CBS^+/+ mice (n = 4) and CBS^–/+ mice (n = 10). ^AP = 0.07.

Figure 6

Cardiovascular histology of CBS^–/+ mouse (Masson trichrome staining). (a) Normal histological aspect of the aorta in a 10-week-old CBS mouse. (b) Cross section through an arteriole in the mesentery of a CBS mouse at 10 weeks of age. Original magnifications: (a) ×120, (b) ×200.

Figure 7

Aortic arch cross sections stained for 3-nitrotyrosine. (a) CBS^+/+ with primary antibody; (b) CBS^+/+ preincubated with peroxynitrite (positive control); (c) CBS^–/+ without primary antibody (negative control); (d) CBS^–/+ with primary antibody.

Figure 8

Aortic arch cross sections stained for eNOS in (a) CBS^+/+ and (b) CBS^–/+ mice.