Objective-Recent studies of bone marrow (BM)-transplanted apoE knockout (apoE Ϫ/Ϫ ) mice have concluded that a substantial fraction of smooth muscle cells (SMCs) in atherosclerosis arise from circulating progenitor cells of hematopoietic origin. This pathway, however, remains controversial. In the present study, we reexamined the origin of plaque SMCs in apoE Ϫ/Ϫ mice by a series of BM transplantations and in a novel model of atherosclerosis induced in surgically transferred arterial segments. Methods and Results-We analyzed plaques in lethally irradiated apoE Ϫ/Ϫ mice reconstituted with sex-mismatched BM cells from eGFP ϩ apoE Ϫ/Ϫ mice, which ubiquitously express enhanced green fluorescent protein (eGFP), but did not find a single SMC of donor BM origin among Ϸ10 000 SMC profiles analyzed. We then transplanted arterial segments between eGFP ϩ apoE Ϫ/Ϫ and apoE Ϫ/Ϫ mice (isotransplantation except for the eGFP transgene) and induced atherosclerosis focally within the graft by a recently invented collar technique. No eGFP ϩ SMCs were found in plaques that developed in apoE Ϫ/Ϫ artery segments grafted into eGFP ϩ apoE Ϫ/Ϫ mice. Concordantly, 96% of SMCs were eGFP ϩ in plaques induced in eGFP ϩ apoE Ϫ/Ϫ artery segments grafted into apoE Ϫ/Ϫ mice. Conclusions-These experiments show that SMCs in atherosclerotic plaques See page 2579 and coverUntil recently, the only source of SMCs in atherosclerotic lesions was considered to be the local vessel wall. According to this hypothesis, local SMCs in the arterial media and intima modulate into a synthetic and migratory phenotype (aka, phenotypic modulation 3 ) and form the fibrous component of the plaque. This theory was essentially inferred from a line of suggestive observations in arterial injury models in the 70s and 80s. 4,5 More to the point, Cre/lox fate mapping in the apoE knockout (apoE Ϫ/Ϫ ) mouse model of atherosclerosis recently confirmed that preexisting SMCs, presumably from the local media, contribute to plaque SMCs during atherogenesis, but the existence of other sources could not be excluded by this technique. 6 In 2002, an alternative and major source of SMCs in atherosclerosis was reported, 7 and this new paradigm has great impact on current research in this area. 8,9 Based on observations in bone marrow (BM)-transplanted apoE Ϫ/Ϫ mice, it was concluded that a substantial fraction of plaque SMCs arise from circulating progenitor cells of hematopoietic origin. 7 This pathway holds promise for the development of novel therapeutic means of controlling the recruitment and accumulation of SMCs in plaques. However, it remains questionable whether the quality of the histological documentation in that study can support a definitive conclusion, especially because the seeming use of unfixed tissue for detection of enhanced green fluorescent protein (eGFP) can lead to diffusion of the tracer marker from sectioned cells. 10 Furthermore, reconstitution of apoE Ϫ/Ϫ mice with apoE To address these concerns, we repeated the BM transplantation experiment in apoE Ϫ/Ϫ mice w...

Abstract. Cardiovascular mortality is 10 to 20 times increased in patients with chronic renal failure (CRF). Risk factors for atherosclerosis are abundant in patients with CRF. However, the pathogenesis of cardiovascular disease in CRF remains to be elucidated. The effect of CRF on the development of atherosclerosis in apolipoprotein E-deficient male mice was examined. Seven-week-old mice underwent 5/6 nephrectomy (CRF, n ϭ 28), unilateral nephrectomy (UNX, n ϭ 24), or no surgery (n ϭ 23). Twenty-two weeks later, CRF mice showed increased aortic plaque area fraction (0.266 Ϯ 0.033 versus 0.045 Ϯ 0.006; P Ͻ 0.001), aortic cholesterol content (535 Ϯ 62 versus 100 Ϯ 9 nmol/cm 2 intimal surface area; P Ͻ 0.001), and aortic root plaque area (205,296 Ϯ 22,098 versus 143,662 Ϯ 13,302 m 2 ; P Ͻ 0.05) as compared with no-surgery mice; UNX mice showed intermediate values. The plaques from uremic mice contained CD11b-positive macrophages and showed strong staining for nitrotyrosine. Systolic BP and plasma homocysteine concentrations were similar in uremic and nonuremic mice. Plasma urea and cholesterol concentrations were elevated 2.6-fold (P Ͻ 0.001) and 1.5-fold (P Ͻ 0.001) in CRF compared with no-surgery mice. Both variables correlated with aortic plaque area fraction (r 2 ϭ 0.5, P Ͻ 0.001 and r 2 ϭ 0.3, P Ͻ 0.001, respectively) and with each other (r 2 ϭ 0.5, P Ͻ 0.001). On multiple linear regression analysis, only plasma urea was a significant predictor of aortic plaque area fraction. In conclusion, the present findings suggest that uremia markedly accelerates atherogenesis in apolipoprotein E-deficient mice. This effect could not be fully explained by changes in BP, plasma homocysteine levels, or total plasma cholesterol concentrations. Thus, the CRF apolipoprotein E-deficient mouse is a new model for studying the pathogenesis of accelerated atherosclerosis in uremia.

Abstract-Hyperhomocysteinemia is an independent risk factor for atherothrombosis. However, causality is unproven, and it remains unknown whether hyperhomocysteinemia promotes atherosclerosis, plaque rupture, and/or thrombosis. We evaluated the short-and long-term effects of hyperhomocysteinemia on plaque size and structure in 99 atherosclerosis-prone apolipoprotein E-deficient mice. Hyperhomocysteinemia was induced by methionine (Met) or homocysteine (HcyH) supplementation: low Met (ϩ11 g Met/kg food), high Met (ϩ33 g Met/kg food), low HcyH (0.9 g HcyH/L drinking water), and high HcyH (1.8 g HcyH/L drinking water). Met and HcyH supplementation significantly raised plasma total homocysteine levels by 4-to 16-fold above those observed in mice fed a control diet (up to 146.1 mol/L). Compared with controls, aortic root plaque size was significantly larger in supplemented groups after 3 months (56% and 173% larger in high-Met and high-HcyH, respectively) but not after 12 months. Hyperhomocysteinemia was associated with an increase in the amount of collagen in plaques after both 3 and 12 months. Mechanical testing of the tail tendons revealed no weakening of collagen after 12 months of hyperhomocysteinemia. Many plaques in both control and supplemented mice appeared rupture prone morphologically, but all aortic root plaques and all but 1 coronary plaque had an intact surface without rupture or thrombosis. Thus, diet-induced hyperhomocysteinemia promotes early atherosclerosis and plaque fibrosis but does not, even in the long term, weaken collagen or induce plaque rupture.

Increased macrophage density in carotid atherosclerotic plaques was associated with lipid content, plaque echolucency, and increased plasma and low-density lipoprotein cholesterol levels. Furthermore, use of aspirin was associated with reduced macrophage density in carotid artery plaques.