Abstract-Blood vessel cells express the 2 known estrogen receptors, ␣ and ␤ (ER␣, ER␤), which are thought to mediate estrogen inhibition of vascular injury and atherosclerosis, but the relative role of ER␣ and ER␤ in these events is controversial. Key Words: estrogen Ⅲ hormones Ⅲ vascular injury Ⅲ receptors Ⅲ animal models T he cardiovascular effects of steroid hormones are an area of intense interest at present. Estrogen has known systemic effects on circulating factors and has more recently been established to have direct effects on the blood vessel wall. Estrogen causes both rapid vascular dilatation and longer-term effects on gene expression in vascular cells (reviewed in Mendelsohn and Karas 1 ). At physiologically relevant concentrations of estrogen, studies support that estrogen receptors (ERs) ␣ and ␤ mediate both the rapid and the long-term cardiovascular effects of estrogen. ER␣ and ER␤ are expressed in both vascular endothelial and smooth muscle cells, but their physiological roles in the vasculature are incompletely understood.Using wild-type and estrogen receptor knockout mice, we have previously studied the role of ER␣ and ER␤ in mediating the vascular protective effects of estrogen in a mouse carotid artery injury model. 2,3,5,6 Studies of mice harboring single gene deletions of either ER␣ or ER␤ showed that treatment of ovariectomized female mice with nanomolar concentrations of 17␤-estradiol (E 2 ) inhibits the response to vascular injury to equivalent levels in wild-type mice, ER␣KO Chapel Hill (ER␣KO CH ) and ER␤KO CH . 2,5 These findings suggested that ER␣ and ER␤ are able to complement one another such that each receptor alone is sufficient to mediate the vascular protective effects of estrogen, or that the vascular protective effects of estrogen are mediated by an ER␣/ER␤-independent pathway. To distinguish between these 2 hypotheses, studies of vascular injury in ER␣,␤KO CH (double) estrogen receptor knockout mice were performed. 6 However, the effect of estrogen on vascular injury in these mice was complex. Although E 2 no longer inhibited the increases in medial carotid area after injury in the ER␣,␤KO CH mice, E 2 still significantly inhibited vascular smooth muscle cell (VSMC) proliferation after injury. In addition, E 2 also caused a significant increase in uterine weight in the ER␣,␤KO CH mice. 6 These data showed that the role of estrogen receptors could diverge for specific components of the vascular injury response in the ER␣,␤KO CH mice. However, the results left unresolved what is responsible for estrogen inhibition of VSMC proliferation and the increase in uterine weight in the ER␣,␤KO CH mice. These could be due to an unidentified third estrogen receptor or to residual function of protein from an ER␣ splice variant known to be expressed in the parental ER␣KO CH mice. 7 To resolve the question as to how estrogen Original

Background and Purpose-We sought to assess the reproducibility, interobserver variability, and application to clinical studies of a new method for the quantitative assessment of carotid plaque echogenicity. Methods-Carotid plaques were scanned with the use of ultrasound, and their images were stored in a computer. They were normalized by assigning certain gray values to blood and adventitia, and the gray scale median (GSM) was used to quantify their echogenicity. The variability between storage media, between degrees of magnification, and between probes was assessed. The method was applied to 232 asymptomatic carotid plaques causing 60% to 99% stenosis in relation to the presence of ipsilateral CT-demonstrated brain infarcts. In all parts of the study the plaque GSM was measured before and after normalization to evaluate its effect. Interobserver agreement for the scanning process was assessed. Results-The GSM mean difference before and after normalization for variability studies of storage media, degrees of magnification, and probes was Ϫ14.5 and Ϫ0.12, 2.24 and 1.68, and Ϫ8.3 and Ϫ0.7, respectively. The median GSM of plaques associated with ipsilateral nonlacunar silent CT-demonstrated brain infarcts was 14, and that of plaques that were not so associated was 30 (Pϭ0.003). The interobserver GSM difference was Ϫ0.05 (95% CI, Ϫ1.7 to 1.6). Conclusions-Our method decreases the variability between storage media and between probes but not the variability between degrees of magnification. It separates echomorphologically the carotid plaques associated with silent nonlacunar CT-demonstrated brain infarcts from plaques that are not so associated.

Abstract-The two known estrogen receptors, ER␣ and ER␤, mediate the effects of estrogen in all target tissues, including blood vessels. We have shown previously that estrogen inhibits vascular injury response to the same extent in female wild-type (WT), ER␣ knockout (ER␣KO CH ), and ER␤ knockout (ER␤KO CH ) mice. We generated mice harboring disruptions of both ER␣ and ER␤ genes (ER␣,␤KO CH ) by breeding and studied the effect of 17␤-estradiol ( Using wild-type (WT) and estrogen receptor knockout (KO) mice, we have previously studied the role of ER␣ and ER␤ in mediating the vascular protective effects of estrogen in a mouse carotid artery injury model. 10,17,18 Previous studies used mice developed at the University of North Carolina, Chapel Hill, which harbor gene deletions of either ER␣ (ER␣KO CH ) 19 or ER␤ (ER␤KO CH ). 20 These studies show that treatment of ovariectomized female mice with nanomolar concentrations of 17␤-estradiol (E2) inhibits the response to vascular injury to equivalent levels in wild-type, 17 ER␣KO CH , 10 and ER␤KO CH mice. 18,21 These findings suggest that ER␣ and ER␤ are able to complement one another such that each receptor alone is sufficient to mediate the vascular protective effects of estrogen, or that the vascular protective effects of estrogen are mediated by an ER␣/ER␤-independent pathway. To distinguish between these two hypotheses, we undertook the present study examining the effect of estrogen on the response to vascular injury in ER␣,␤KO CH (double) estrogen receptor knockout mice. Materials and Methods AnimalsA total of over 820 animals were required to ultimately generate the ER␣,␤KO CH mice used in this study. These mice, which have been extensively studied, do not express ER proteins in any tissue. 19,20,[22][23][24] However, ER␣KO CH mice have been shown previously to express mRNA for 2 partial ER␣ transcripts, one of which retains the hormone-and DNA-binding domains of full-length ER␣, and can Original