Estradiol alters nitric oxide production in the mouse aorta through the alpha-, but not beta-, estrogen receptor.

Although estradiol (E(2)) has been recognized to exert several vasculoprotective effects in several species, its effects in mouse vasomotion are unknown, and consequently, so is the estrogen receptor subtype mediating these effects. We investigated the effect of E(2) (80 microg/kg/day for 15 days) on NO production in the thoracic aorta of ovariectomized C57Bl/6 mice compared with those given placebo. E(2) increased basal NO production. In contrast, the relaxation in response to ATP, to the calcium ionophore A23187, and to sodium nitroprusside was unaltered by E(2), whereas acetylcholine-elicited relaxation was decreased. The abundance of NO synthase I, II, and III immunoreactive proteins (using Western blot) in thoracic aorta homogenates was unchanged by E(2). To determine the estrogen receptor (ER) subtype involved in these effects, transgenic mice in which either the ERalpha or ERbeta has been disrupted were ovariectomized and treated, or not, with E(2). Basal NO production was increased and the sensitivity to acetylcholine decreased in ERbeta knockout mice in response to E(2), whereas this effect was abolished in ERalpha knockout mice. Finally, these effects of E(2) on vasomotion required long-term and/or in vivo exposure, as short-term incubation of aortic rings with 10 nmol/L E(2) in the isolated organ chamber did not elicit any vasoactive effects. In conclusion, this study demonstrates that ERalpha, but not ERbeta, mediates the beneficial effect of E(2) on basal NO production.

The AF-1 activation-function of ERalpha may be dispensable to mediate the effect of estradiol on endothelial NO production in mice.

Two isoforms of estrogen receptor (ER) have been described: ERalpha and ERbeta. The initial gene targeting of ERalpha, consisting in the introduction of a Neo cassette in exon 1 [alphaERKO, hereafter called ERalpha-Neo KO (knockout)], was reported in 1993. More recently, another mouse deficient in ERalpha because of the deletion of exon 2 (ERalphaKO, hereafter called ERalpha-delta2 KO) was generated. In ovariectomized ERalpha-wild-type mice, estradiol (E(2)) increases uterine weight and basal production of endothelial nitric oxide (NO). Both of these effects are abolished in ERalpha-delta2 KO mice. In contrast, we show here that both of these effects of E(2) are partially (uterine weight) or totally (endothelial NO production) preserved in ERalpha-Neo KO. We also confirm the presence of two ERalpha mRNA splice variants in uterus and aorta from ERalpha-Neo KO mice. One of them encodes a chimeric ERalpha protein (ERalpha55), partially deleted in the A/B domain, that was detected in both uterus and aorta by Western blot analysis. The other ERalpha mRNA splice variant codes for an isoform deleted for the A/B domain (ERalpha46), which was detected in uterus of ERalpha-Neo KO, and wild-type mice. This protein isoform was not detected in aorta. The identification of these two N-terminal modified isoforms in uterus, and at least one of them in aorta, probably explains the persistence of the E(2) effects in ERalpha-Neo KO mice. Furthermore, ERalpha-Neo KO mice may help in the elucidation of the specific functions of full-length ERalpha (ERalpha66) and ERalpha46, both shown to be physiologically generated in vivo.

Omapatrilat, a dual angiotensin-converting enzyme and neutral endopeptidase inhibitor, prevents fatty streak deposit in apolipoprotein E-deficient mice.

Angiotensin-converting enzyme (ACE) is mainly responsible for converting angiotensin I (AI) to angiotensin II (AII), and ACE inhibitors prevent atherosclerosis in animal models. Neutral endopeptidase 24.11 (NEP) degrades substance P, kinins and atrial natriuretic peptide (ANP), and aortic wall NEP activity was found to be increased in atherosclerosis. In the present study, we have evaluated the effect of candoxatril, a NEP inhibitor, and of omapatrilat, a dual ACE and NEP inhibitor, on the development of fatty streak in apolipoprotein E (apoE)-deficient mice. Groups of ten male apoE-deficient mice were given either placebo, candoxatril 50 mg/kg per day, or omapatrilat 10, or 100 mg/kg per day for 4 months. None of the treatments influenced body weight, serum total or HDL-cholesterol. Compared with the placebo, candoxatril did not protect the mice from fatty streak deposit. In contrast, omapatrilat dose dependently inhibited the constitution of fatty streak in apoE-deficient mice. The precise advantages of the dual ACE and NEP inhibition versus the inhibition of only ACE should now be considered in the prevention of atherosclerosis as well as in the occurrence of its complications.

[Effect of estrogens on arterial physiology and mechanisms of their atherosclerosis-protective effect in animals]. / Effect des oestrogènes sur la physiologie artérielle et mécanismes de son effet athéroprotecteur chez l'animal.

Two isoforms of oestrogen receptors (alpha and beta) have been identified in the cells of the arterial wall, and a heterogeneity of their expression according to the animal species, the vascular beds and the sex has been reported. Oestrogens can thus directly influence the vascular physiology through a 'genomic' mechanisms, but 'extra-genomic' mechanisms responsible for a short-term effect have also been suggested. Oestrogens potentiate endothelium-dependent relaxation through an increase in nitric oxide bioavailability (increase in its production and/or decrease in its degradation by superoxide anion according to the vascular beds). Endothelial 'dysfunction' (abnormality of the endothelium-dependent vasodilation) occurs in atheromatous arteries. Oestrogen replacement prevents and even corrects this endothelial dysfunction. In monkeys, this beneficial effect of oestrogens is not altered by coadministration of progesterone, but is abolished by coadministration of medroxyprogesterone. Finally, oestrogens prevent fatty streak deposit, and the mechanisms of this atheroprotective effect are being studied.

Endothelium-derived nitric oxide and vascular physiology and pathology.

In 1980, Furchgott and Zawadzki demonstrated that the relaxation of vascular smooth muscle cells in response to acetylcholine is dependent on the anatomical integrity of the endothelium. Endothelium-derived relaxing factor was identified 7 years later as the free radical gas nitric oxide (NO). In endothelium, the amino acid L-arginine is converted to L-citrulline and NO by one of the three NO synthases, the endothelial isoform (eNOS). Shear stress and cell proliferation appear to be, quantitatively, the two major regulatory factors of eNOS gene expression. However, eNOS seems to be mainly regulated by modulation of its activity. Stimulation of specific receptors to various agonists (e.g., bradykinin, serotonin, adenosine, ADP/ATP, histamine, thrombin) increases eNOS enzymatic activity at least in part through an increase in intracellular free Ca2+. However, the mechanical stimulus shear stress appears again to be the major stimulus of eNOS activity, although the precise mechanisms activating the enzyme remain to be elucidated. Phosphorylation and subcellular translocation (from plasmalemmal caveolae to the cytoskeleton or cytosol) are probably involved in these regulations. Although eNOS plays a major vasodilatory role in the control of vasomotion, it has not so far been demonstrated that a defect in endothelial NO production could be responsible for high blood pressure in humans. In contrast, a defect in endothelium-dependent vasodilation is known to be promoted by several risk factors (e.g., smoking, diabetes, hypercholesterolemia) and is also the consequence of atheroma (fatty streak infiltration of the neointima). Several mechanisms probably contribute to this decrease in NO bioavailability. Finally, a defect in NO generation contributes to the pathophysiology of pulmonary hypertension. Elucidation of the mechanisms of eNOS enzyme activity and NO bioavailability will contribute to our understanding the physiology of vasomotion and the pathophysiology of endothelial dysfunction, and could provide insights for new therapies, particularly in hypertension and atherosclerosis.