The present study was designed to test the hypothesis that estrogen receptors (ER) in the blood vessel wall play a role in the modulation of the release of endothelium-derived nitric oxide (EDNO). Both basal and stimulated release of EDNO were determined in aortic rings isolated from female and male wild-type and male homozygous estrogen receptor knock-out (ERKO) mice.125 I-17 ␤ -estradiol binding in aortic tissue showed significantly more high affinity cytosolicnuclear-binding sites in male compared with female wildtype mice. Estrogen receptor transcripts were present in the aorta of male wild-type mice, but they were absent in male ERKO animals. Basal release of EDNO (determined by endothelium-dependent contraction caused by N G -nitro-Larginine) was significantly higher in aorta of wild-type male mice compared with wild-type female mice, and significantly lower in the aorta of male ERKO compared with male wild-type mice. Acetylcholine-induced endotheliumdependent relaxation was similar in all groups studied. No difference was observed in the activity of calcium-dependent nitric oxide synthase in homogenates of lungs and brain taken from male wild-type and ERKO mice. These studies show a significant association between the number of estrogen receptors and basal release of EDNO in the aorta of mice, and suggest that decreased vascular estrogen receptor number may represent a novel risk factor for cardiovascular diseases.

Abstract-We have previously demonstrated that urokinase-type plasminogen activator (uPA) is highly expressed in the aneurysmal segment of the abdominal aorta (AAA) in apolipoprotein E-deficient (apoE Ϫ/Ϫ ) mice treated with angiotensin II (Ang II). In the present study, we tested the hypothesis that uPA is essential for AAA formation in this model. An osmotic minipump containing Ang II (1.44 mg/kg per day) was implanted subcutaneously into 7-to 11-month-old male mice for 1 month. Ang II induced AAA in 9 (90%) of 10 hyperlipidemic mice deficient in apoE (apoE Ϫ/Ϫ /uPA ϩ/ϩ mice) but in only 2 (22%) of 9 mice deficient in both apoE and uPA (apoE Ϫ/Ϫ /uPA Ϫ/Ϫ mice) (PϽ0.05). Although the expansion of the suprarenal aorta was significantly less in apoE Ϫ/Ϫ /uPA Ϫ/Ϫ mice than in apoE Ϫ/Ϫ /uPA ϩ/ϩ mice, the aortic diameters of the aorta immediately above or below the suprarenal aorta were similar between the 2 groups. Ang II induced AAA in 7 (39%) of 18 strain-matched wild-type C57 black/6J control mice. The incidence was significantly higher in atherosclerotic apoE-deficient (apoE Ϫ/Ϫ ) mice, in which 8 (100%) of 8 mice developed AAA. Only 1 (4%) of 27 uPA Ϫ/Ϫ mice developed AAA after Ang II treatment. We conclude the following: (1) uPA plays an essential role in Ang II-induced AAA in mice with or without preexisting hyperlipidemia and atherosclerosis; (2) uPA deficiency does not affect the diameter of the nonaneurysmal portion of the aorta; and (3)

Urokinase-type plasminogen activator (uPA) is increased in human abdominal aortic aneurysm (AAA).Chronic infusion of angiotensin II (Ang II) results in AAA in apolipoprotein E-deficient mice. We tested the hypothesis that Ang II infusion results in an elevation of uPA expression contributing to aneurysm formation. Ang II or vehicle was infused by osmotic pumps into apoE-KO mice. All mice treated with Ang II developed a localized expansion of the suprarenal aorta (75% increase in outer diameter), accompanied by an elevation of blood pressure (22 mmHg), compared to the vehicle-treated group. Histological examination of the dilated aortic segment revealed similarities to human AAA including focal elastin fragmentation, macrophage infiltration, and intravascular hemorrhage. Ang II treatment resulted in a 13-fold increase in the expression of uPA mRNA in the AAA segment in contrast to a twofold increase in the atherosclerotic aortic arch. Increased uPA protein was detected in the abdominal aorta as early as 10 days after Ang II infusion before significant aorta expansion. Thus, Ang II infusion results in macrophage infiltration, increased uPA activity, and aneurysm formation in the abdominal aorta of apoE-KO mice. These data are consistent with a causal role for uPA in the pathogenesis of AAA. Abdominal aortic aneurysm (AAA) is a chronic degenerative disease characterized by segmental weakening and dilation of the vascular wall. Recent estimates indicate that the prevalence of AAA is 4 to 9% in adults older than 65 years of age and is known to be associated with atherosclerosis, aging, hypertension, and cigarette smoking. Continued tissue remodeling results in silent expansion of the AAA with an increased risk of spontaneous rupture. Currently the only available treatments for AAAs are surgical resection and replacement or, more recently, insertion of an endovascular stent. The etiology of AAA is unclear. The extracellular matrix plays an essential role in maintaining the integrity of the vascular wall. Elastin and collagen fibers are the major components of this extracellular matrix. Both plasmin and matrix metalloproteinases (MMPs) are capable of degrading extracellular matrix, including collagen, elastin, and fibrin. Urokinase-type plasminogen activator (uPA) hydrolyzes plasminogen to form plasmin, which in turn activates MMPs. The in vivo activity of uPA is also regulated by local concentrations of its major inhibitor, PAI-1. Biochemical studies have demonstrated increased proteolytic activity in the aortic wall of AAA. Schneiderman and colleagues 1 showed that uPA mRNA as well as the tissue-type plasminogen activator (tPA), co-localized with infiltrating macrophages, is significantly increased in human AAA. Increased activities of MMP-2, -3, -9, and -12 in AAA have also been reported. [2][3][4][5] Atherosclerotic aortic lesions from high-cholesterol diet-fed apoE-KO mice show fragmentation of the elastic lamellae and rupture of the media resulting in pseudomicroaneurysm formation. These pathological changes are ...

Atherosclerosis develops and progresses spontaneously in apolipoprotein E-knockout (apoE-KO) mice. A direct consequence of atherosclerosis is an increase in vascular stiffness. Pulse wave velocity (PWV) has been used to assess the stiffness of large vessels and was found to be increased in patients with atherosclerosis. In the present study, aortic stiffness was assessed by PWV in 4- and 13-mo-old apoE-KO mice and age-matched controls (C57BL/6J). In 13-mo-old apoE-KO mice with extensive atherosclerotic lesions in the aorta (61 ± 4%), PWV increased significantly (3.8 ± 0.2 m/s) compared with controls (2.9 ± 0.2 m/s). Endothelial nitric oxide (EDNO)-mediated vasorelaxation in response to ACh was markedly diminished in the aortic rings isolated from 13-mo-old apoE-KO mice compared with age-matched controls. In contrast, in 4-mo-old apoE-KO mice with only moderate atherosclerotic lesions in the aorta (23 ± 5%), there were no significant changes in PWV and EDNO-mediated relaxation compared with controls. Blood pressure was not different among the four groups of mice. There were no significant differences in endothelium-independent vascular responses to sodium nitroprusside among different groups investigated. Histological evaluation revealed focal fragmentation of the elastic laminae in the aortic walls of 13-mo-old apoE-KO mice. These results demonstrate for the first time that aortic stiffness determined by PWV increases in 13-mo-old apoE-KO mice. Endothelial dysfunction and elastic destruction in vascular wall caused by atherosclerosis may have contributed.

Mechanoreception, a widely distributed sensory modality, has been shown to be present in certain blood vessels. Changes in physical forces, like sudden increase of transmural pressure or flow velocity (shear stress), trigger changes in blood vessel diameter; the former reduces it while the latter increases vessel caliber. These changes in diameter, which are the result of contraction and relaxation of vascular smooth muscle in the blood vessel media, can serve the purpose of physiological regulation of blood flow (autoregulation) and protection of the intima against damages from high shear forces. The precise location of mechanosensor(s) and the mechanism of mechanoreception and signal transduction are poorly understood. Accumulating evidence suggests that the endothelium may be a site of mechanoreception and that changes in the synthesis/release of endothelium-derived relaxing (EDRF, EDHF, PGI₂) and contracting factors (EDCF) result in altered vascular smooth muscle tone and vessel caliber. Increased shear stress stimulates the release of EDRF and PGI2 probably via activation of a K⁺ channel (inward rectifier) in endothelial cell membrane. Endothelium-dependent vascular contraction evoked by increased transmural pressure may be the result of (1) reduced release of EDRF (canine carotid artery) and (2) stimulation of the release of a still unidentified EDCF(s) (feline cerebral artery). Thus the endothelium can serve as pressure and flow sensor and is capable of transducing changes in mechanical forces into changes of vascular smooth muscle tone by modulating the release of endothelium-derived vasoactive factors. The physiological importance of the mechanoreception by endothelial cells in the intact circulation remains to be determined.

17 beta-Estradiol induces vasodilation in vitro and in vivo, which has been suggested to contribute to the cardiovascular protection by this ovarian steroid hormone. However, the exact mechanism of vasorelaxation by estrogens remains to be elucidated. In this study, we analyzed the potential role of genomic mechanisms involving the nuclear estrogen receptor and inhibition of entry of extracellular Ca2+ in 17 beta-estradiol-induced vasorelaxation in depolarized aortic rings, isolated from male and female rats and male mice. In both male and female rat aortic rings without endothelium and in intact male mouse aortic rings treated with NG-nitro-L-arginine, 17 beta-estradiol caused dose-dependent (0.3 to 30 mumol/L) relaxation of contraction evoked by high-K+ depolarization (30 and 45 mmol/L KCl, respectively). The estrogen receptor antagonist ICI 164384 had no effect on 17 beta-estradiol-induced relaxations. 125I-17 beta-estradiol binding studies showed the presence of high-affinity cytosolic-nuclear estrogen receptors in control male mouse aortas. Comparable relaxations of aortic rings isolated from control and estrogen receptor-deficient transgenic mice provided direct evidence that the nuclear estrogen receptor is not involved in this response. 17 beta-Estradiol-induced relaxation of rat aortic rings could not be prevented by cycloheximide or actinomycin D, suggesting that the response was not mediated by de novo protein synthesis or gene transcription. In rat aortic rings, 17 beta-estradiol inhibited the increase of 45Ca uptake by 30 mmol/L KCl at concentrations (10 and 30 mumol/L) that caused vasorelaxation in the same tissue, suggesting that inhibition of Ca2+ entry contributes to the response. 17 alpha-Estradiol was less effective, and estrone was devoid of vasorelaxing activity. Vasorelaxation by estrogens in female and male rat aortas was similar, indicating no gender difference in vascular responses under these conditions.

Estrogen is known to modulate angiogenesis, both under physiological and pathological conditions, and has been demonstrated to augment angiogenesis induced by bFGF in a mouse model. We have modified this mouse model and measured the apparent plasma volume in Matrigel plugs containing basic fibroblast growth factor (bFGF) in wild type and estrogen receptor knockout, ovariectomized mice in the presence and absence of exogenous 17 beta estradiol. The apparent plasma volume was determined by measuring the fluorescence of the excised plug 10 min. after injection of fluoroscein labeled dextran 150. In wild type mice exogenous 17 beta estradiol increased the apparent plasma volume of the Matrigel plug and the uterine weight significantly. In the estrogen receptor knockout mice exogenous 17 beta estradiol caused a small, but significant increase in uterine weight but was without effect on the apparent plasma volume of the Matrigel plug. It is concluded that functional estrogen receptors are essential for the augmentation of bFGF-induced angiogenesis by exogenous 17 beta estradiol in female mice.

The relative importance of intracellular and extracellular Ca2+ in the release of endothelium-derived relaxing factor (EDRF) and the mechanisms involved in the release of intracellular Ca2+ were investigated in cultured bovine endothelial cells. The release of EDRF by bradykinin, determined by bioassay, was dose-dependent showing an EC50 of 4 x 10(-10) M. The bradykinin-induced EDRF release from endothelial cells was maintained in the presence of extracellular Ca2+. However, in the absence of external Ca2+, bradykinin-induced EDRF release was both attenuated and transient. In cells loaded to isotopic equilibrium with 45Ca, bradykinin increased the 45Ca efflux into both calcium-containing and calcium-free solutions, with an EC50 for the increase in 45Ca efflux induced by bradykinin of 1.3 x 10(-9) M. The involvement of an intracellular Ca2+ store and the participation of a second messenger in its release were investigated in saponin-permeabilized endothelial cells. In saponin-permeabilized cells, ATP-sensitive calcium uptake was Ca2+,Mg2+ -ATPase-dependent. The ATP-sensitive uptake of calcium at different free Ca2+ concentrations showed at least two compartments involved in the uptake of Ca2+. The 45Ca uptake into the compartment with the lowest affinity and highest capacity could be inhibited by sodium azide, suggesting that this uptake was into mitochondria. The majority of the 45Ca uptake into the azide-insensitive store could be released by inositol-1,4,5-trisphosphate (IP3). The IP3-induced release was not affected by apyrase or exogenous GTP. The EC50 for the release of Ca2+ by IP3 was 1.0 microM and was unaffected by an inhibitor of IP3 breakdown (2,3-diphosphoglyceric acid).(ABSTRACT TRUNCATED AT 250 WORDS)