The mechanism by which Hey may contribute to vascular damage is an active field of investigation. Damage to endothelium appears to be a key early event in many vascular diseases^ ^ and may play a central role in the promotion of atherosclerosis by Hey. The endothelium is a monolayer of cells sited at the interface between blood and vessel wall forming a continuous lining throughout the vascular tree. Endothelium-derived relaxing factor (EDRF), shown to be nitric oxide (NO), is synthesized from L-arginine within the endothelial cell by the constitutive enzyme nitric oxide synthase^^. NO is released fi'om the endothelium in response to various stimuli of which an important physiological stimulus is pulsatile flow. NO plays a vital role in controlling vascular reactivity, vascular remodelling and coagulation. Endothelial dysfunction, leading to reduced NO activity, may occur without histological evidence of endothelial damage or significant atheroma^^.
The ‘response to injury hypothesis’ identifies endothelial injury as a critical initiating event in the development of atherosclerosis^^. Following endothelial damage, proliferation of smooth muscle cells, increased synthesis of sulphated proteoglycosaminoglycans, fibrosis, calcification and deposition of lipoprotein are important processes in the development of atherosclerosis. Homocysteine could potentially be involved in several of these processes. Indeed, a variety of possible mechanisms for Hcy-mediated vascular injury have been proposed and are reviewed below.
1.7.1 Cellular toxicity and endothelial injury
Homocysteine may exert an effect by direct toxicity to any cellular component of the arterial wall. The effect of Hey on endothelial function has been studied in various ways. Studies using endothelial cells in vitro have shown evidence of cellular injury in the presence of Hey. Thus Hey can induce cellular damage as indicated by ^^Cr release from labelled human endothelial cell monolayers in a dose-dependent manner^^. However, the significance of these data must be questioned in that the effect is not specific to Hey as it has been shown by others that cysteine can also induce the detachment of endothelial cells at the concentrations used (ca. 5mM)^^.
Several studies in vivo have demonstrated endothelial abnormalities following Hey infusion. One such study^^, demonstrated endothelial desquamation in baboons infused with Hey thiolactone for 5 days. Sustained infusion (3 months) produced eccentric fibromuscular lesions containing intracellular lipids and foam cells, resembling early atherosclerotic lesions in humans.
Proliferation of vascular smooth muscle cells is a prominent feature of atherosclerosis^^. This is another possible mechanism for Hcy-induced vascular injury. In support of this proposal Hey has been shown to have a direct growth promoting effect on vascular smooth muscle cells in culture^^.
The function of endothelium and vascular smooth muscle are intimately related in several respects including the control of vascular tone and blood flow. NO released by endothelium in response to pulsatile flow^® acts locally to produce
may therefore affect these functions. The bioavailability of nitric oxide may be decreased by reacting with reactive oxygen species such as superoxide, or its activity may be conserved in the presence of reduced thiols forming nitrosothiols. S- nitrosothiols have potent vasodilatory and anti-platelet effects^ ^ In the presence of Hey, nitric oxide forms S-nitrosohomocysteine.
Adverse effects of Hey may occur when the endothelium is unable to support nitrosothiol formation due to endothelial injury. Under these conditions. Hey may then generate H2O2 and undergo conversion to Hcy-thiolactone, which may further
contribute to endothelial dysfunction. Homocysteine-mediated endothelial cell toxicity has been shown to be dependent on the duration of exposure to Hcy^^, brief (15 min) exposure of endothelial cells to Hey resulted in the formation of S- nitrosohomocysteine whereas prolonged (>3 hrs) exposure resulted in impaired NO- mediated responses.
Results, however, from in vitro experiments need to be interpreted with caution. Most have used high concentrations of Hey well above the range for mild hyperhomocysteinaemia and even homocystinuria. Lentz and co-workers have recently reported the effect of more relevant levels of Hey on endothelial function^^. This primate model demonstrated vascular dysfunction in monkeys with hyperhomocysteinaemia, achieving an increase in plasma tHcy from 4 to lOpM by dietary modification (methionine feeding, folate deficient). In vivo changes in blood flow to the leg after infusion of endothelium-dependent and-independent agonists together with vasomotor responses measured ex vivo in carotid arteries were determined. Relaxation to endothelium-dependent vasodilators was impaired in the hyperhomocysteinaemic monkeys as was the endothelium-independent response to nitroprusside, although to a lesser extent. These data suggest that endothelial function and possibly also smooth muscle cell function may be impaired by mild hyperhomocysteinaemia.
The most persuasive evidence is likely to come from studies in man. Confirmation that endothelial dysfunction occurs in homocystinuria comes from a clinical study by Celermajer and colleagues using non-invasive external vascular ultrasound^. Flow-mediated dilatation of the brachial arteiy is endothelium- dependent and is a measure of the flow-related NO response in that artery^^. In this study, brachial or femoral artery responses to reactive hyperaemia (flow-mediated, endothelium-dependent) were measured in 9 children (4 to 17 yrs) with homozygous
homocystinuria. Although plasma homocysteine concentrations were not measured, flow-related endothelial function was markedly impaired in these children but not in their heterozygous parents or normal controls. These data, if confirmed with other studies, would strongly support the proposal that hyperhomocysteinaemia can cause endothelial damage in humans.
1.7.2 Free-radical mediated damage
It has been proposed by many authors that a molecular mechanism for Hcy- mediated damage may involve free radical production^^. At the simplest level of redox reactions it should be noted that Hey in plasma can exist in its reduced form but is usually rapidly auto-oxidized to homocystine, homocysteine-mixed disulphide and homocysteine-thiolactone. Olszewski and McCully have proposed chemical reactions whereby Hey could lead to production of free radicals^^: -