Angiotensin-converting enzyme 2 (ACE2) is a novel enzyme with possible implications in the treatment of blood pressure disorders. Recent evidence suggests that an upregulation of ACE2 can be stimulated by all-trans retinoic acid (at-RA); however, at-RA also affects regulation of the stem-cell marker octomer-4 (Oct-4) and thus cellular differentiation. We have previously shown that smooth muscle cells and macrophages present within rabbit atherosclerotic plaques are positive for ACE2, Oct-4 and the haematopoietic stem-cell marker CD34. Thus, to provide evidence that possible at-RA treatment could affect both plaque cellular biology (via effects on cellular differentiation) and blood pressure (via ACE2), it is vital to show that cells with atherosclerotic plaques co-express all three markers. Thus, we sought to provide evidence that a subset of cells within atherosclerotic plaques is positive for ACE2, Oct-4 and CD34. We used New Zealand White rabbits that were fed a control diet supplemented with 0.5% cholesterol plus 1% methionine for 4 weeks and then allowed to consume a normal diet for 10 weeks. Immunohistochemistry was performed by standard techniques. We report that ACE2, Oct-4 and CD34 were all present within atherosclerotic plaques. Although macrophages were positive for all three markers, spindle-shaped cells in the media did not show all three markers. The endothelium overlying normal arterial wall showed positive Oct-4 and ACE2 immunoreactivity, but CD34 immunoreactivity was patchy, indicating that such cells might not have fully differentiated. It is concluded that cells in atherosclerotic plaques express co-express ACE2, Oct-4 and CD34. Further studies aimed at establishing the effects of all-trans retinoic acid on blood pressure and atherosclerotic cell differentiation are warranted.

A physiological atherogenic human diet consists of 0.1% cholesterol, fat, as well as high levels of methionine, which is the precursor to homocysteine. The pathological effects of a diet enriched with physiologically high levels of cholesterol, methionine and fat over a short period on the aorta are unknown. In this regard, we sought to determine the effects of a 0.1% cholesterol diet in combination with a 1% methionine over a 4-week period on endothelial function and artery pathology and the expression of endothelial nitric oxide synthase as well as nitrosative stress by nitrotyrosine (NT), oxidative stress by heat shock protein 70 (HSP70) and endoplasmic reticulum stress by glucose regulated protein 78 (GRP78). Rabbits were fed for 4 weeks a diet supplemented with 1% methionine + 0.1% cholesterol + 5% peanut oil (MC). The endothelial function of the abdominal aorta was examined using organ bath techniques, atherosclerosis determined in each artery by microscopy and eNOS, NT, GRP78 and HSP70 by standard immunohistochemistry. Endothelium dependent relaxation in response to acetylcholine significantly decreased by 63% at 1 muM acetylcholine (P < 0.001) compared with control arteries. There was no evidence of atherosclerosis formation in any artery studied, however, eNOS, NT and GRP78 was clearly present in all arteries studied but HSP70 was not easily detectable. Severe endothelial dysfunction is present in the abdominal aorta of rabbits within 4 weeks of physiological dietary manipulation, possibly due to NT formation and endoplasmic reticulum stress. This model could be used to study the early onset of endothelial dysfunction prior to the initiation of atherosclerosis.

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