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knockout mice
Karolinska Institutet, 1Institution for Clinical Science, Intervention, and Technology, Division of Obstetrics and Gynecology and 3Department of Medical Nutrition, Karolinska University Hospital, Huddinge, Stockholm, Sweden; and 2Maternal and Fetal Research Unit, Division of Reproductive Health, Endocrinology, and Development, Kings College London, London, United Kingdom
Submitted 1 August 2005 ; accepted in final form 19 September 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-deficient mice (-ERKO) and to gain insight into the role of nitric oxide (NO) in these responses. Femoral arteries (
250 µm) from male and female -ERKO mice and wild-type (WT) littermates (26 female, 13 in each group; and 24 male, 12 in each group) were mounted on a Multi-Myograph. Concentration-response curves to 17-estradiol (17-E2) and the selective estrogen receptor-
(ER-) agonist propyl-[1H]-pyrazole-1,3,5-triy-trisphenol (PPT) were obtained before and after NO synthase (NOS) inhibition [N
-nitro-L-arginine methyl ester (L-NAME), 0.1 mM] in arteries preconstricted with U-46619 (a thromboxane analog). In WT mice, responses to the potent estrogen receptor- (ER-) agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and the contribution of NO were also assessed. Concentration-response curves to 17-E2 and PPT were similar in arteries from WT and -ERKO mice of both genders, but NO-mediated relaxation was different, since L-NAME reduced 17-E2 mediated relaxation in arteries from male and female -ERKO but not WT mice (P < 0.05). NOS inhibition reduced dilation to PPT in arteries from male and female WT mice, as well as arteries from female -ERKO mice (P < 0.05). Responses to DPN in arteries from WT female and male mice did not differ after NOS inhibition. The acute dilatory responses to estrogenic compounds are similar in WT and -ERKO mice but differ mechanistically. Because NO appeared to contribute to responses to 17-E2 in arteries from -ERKO but not WT mice, the presence of ER- apparently inhibits ER--mediated NO relaxation.
femoral arteries; estrogen receptor knockout mice; vasodilation; propyl-[1H]-pyrazole-1,3,5-triy-trisphenol; nitric oxide
and ER- are reported to be present in the plasma membrane of vascular endothelial cells and to be associated with numerous signal transduction pathways, including activation of endothelial nitric oxide synthase (eNOS; see Refs. 6 and 9).
The finding that 17-estradiol (17-E2) administration results in rapid vasodilation, attributed to a plasma membrane ER, has provoked interest in the potential role of nongenomic activation of the ER in vascular homeostasis (8, 32). Nitric oxide (NO) has been implicated, but its role remains controversial, since both NO-dependent and -independent dilatation has been reported in ex vivo studies using isolated arteries (48, 51).
ER- and ER- are expressed in a wide range of blood vessels from different vascular beds and different species (53). However, the prevalence of each ER subtype within the vascular wall, the relative roles of plasma membrane and nuclear receptors, and how the receptor subtypes interact remain uncertain. The observation that the aorta from ER- knockout (-ERKO) male mice demonstrates enhanced dilatation to 17-E2 (40) compared with the response in wild-type (WT) animals could indicate a modulator role of ER- on ER-, suggesting that the relative expression of subtypes of ER present in a given artery may have important consequences for ER signaling and vascular tone.
The present study was carried out using isolated small femoral arteries from female and male -ERKO mice and WT controls. In the WT, these arteries similarly express ER- and ER-, as detected and reported by our group recently, and ER- is absent in these arteries from the -ERKO mice (30). The choice of vasculature was also supported by studies in femoral arteries from rats in which dilatory responses to estrogens were sex specific and, at least in part, NO mediated (51).
We determined whether disruption of ER- alters the acute dilatory response to nanomolar-micromolar concentrations of 17-E2 (nonselective ER agonist; see Ref. 17) and propyl-[1H]-pyrazole-1,3,5-triyl-trisphenol (PPT; ER- selective agonist) (52) and if NO contributes to these responses. In addition, we investigated the vascular responses to the potent ER- agonist 2,3-bis-4-hydroxyphenyl (DPN; see Ref. 33) in arteries from WT mice.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Experiments were performed on age-matched (14–22 wk old) male and female WT mice (WT, ER-; +/+, C57BL/6J) and homozygous mutant mice lacking the gene for ER- (ER-; –/–; see Ref. 21). Mice lacking ER- developed normally and were indistinguishable grossly and histologically as young adults from WT littermates (21). Mice were housed in climate-controlled rooms, and standard rodent chow and water were available ad libitum. Mice were killed by asphyxiation by a rising concentration of CO2. Guidelines were followed for the care and use of experimental animals as issued by Stockholm's Södra Djurförsöksetisk Nämnd and the Institute for Laboratory Animal Research Guide for Care and Use of Laboratory Animals.
Assessment of Artery Function
Femoral arteries (first and second order, 200 µm diameter) were immersed in physiological salt solution (PSS) and freed of adherent connective tissue by microscope-guided dissection, cut to a length 2 mm, and immediately mounted (using 25-µm tungsten wire) on a Multi-Myograph (model 610; Danish Myo Technology). Vessels from male (n = 13) and female (n = 13) -ERKO mice and male (n = 12) and female (n = 12) WT mice were studied. Arteries were maintained in PSS at 37°C, while being continuously oxygenated with 5% CO2 in oxygen. All solutions, including the incubation solutions, were refreshed every 30 min. A standardized normalization procedure was performed to allow calculation of the artery diameter at which the in vivo transmural pressure of relaxed artery would have been 100 mmHg (18). Arteries were set to 80% of this diameter, since preliminary experiments found this to be the optimum resting tension. Commercially available software was used for calibrations and for data collection (Myodac version 2.1; Danish Myo Technology).
After normalization, arteries were left to equilibrate for 20 min. Viability was assessed by contraction to high-potassium (125 mmol/l) and norepinephrine (NE; 1 µmol/l) solution and dilatation to ACh (1 µmol/l). Arteries that failed to achieve contraction equivalent to 13.3 kPa or that demonstrated <60% relaxation to ACh were discarded.
Experimental Protocols
In WT and -ERKO arteries, cumulative concentration-response curves to 17-E2 and to PPT (0.01–10 µM, with additions every 5 min) were obtained (in separate arteries for each agonist) after preconstriction with a submaximal (80%, 0.1 µM) concentration of U-46619 (a thromboxane analog) before and after incubation with the nitric oxide synthase (NOS) inhibitor N-nitro-L-arginine methyl ester (L-NAME; 0.1 mM, 30 min). Responses to the ER- agonist DPN were also evaluated in arteries from WT mice (0.01–10 µM, with additions every 5 min; Fig. 1). The concentration-response curves to each agonist before and after incubation with L-NAME were obtained in the same artery. Pilot studies carried out before the experiments demonstrated that incubation with L-NAME did not influence the basal tension or U-46619 preconstriction, and the second concentration-response curve to the substances after 30 min incubation with PSS alone (without L-NAME) was similar to that obtained before the incubation. Moreover, time controls in which constriction was maintained over the same period but without additions of estrogenic compounds or inhibitors demonstrated that U-46619 preconstriction was stable over the time period of the addition of cumulative concentrations.
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The composition of PSS was as follows (mmol/l): 119 NaCl, 4.7 KCl, 2.5 CaCl2, 21.17 MgSO4, 25 NaHCO3, 1.18 KH2PO4, 0.026 EDTA, and 5.5 glucose, pH 7.4. NE was dissolved in PSS, and both ACh and L-NAME were dissolved in distilled water. PPT, DPN, 17-E2, and U-46619 were dissolved in 95% ethanol. All chemicals were obtained from Sigma-Aldrich (Stockholm, Sweden) with the exception of PPT, DPN, and U-46619, which were obtained from Tocris Cookson (Bristol, UK). The final bath concentration of the ethanol did not exceed 0.01%. Vehicle control concentration-response curves were carried out by adding ethanol to PSS to ensure that the concentration used did not affect U-46619-mediated constriction.
Data Analysis
Tension (mN/mm of artery segment) was calculated using Myodata software (Danish Myo Technology). All measurements were corrected for baseline tension. Data were transferred to STATISTICA (version 6.0; StatSoft), in which all analyses were performed. Relaxation to vasodilators was calculated as percent reduction of U-46619 preconstriction. Data are expressed as means ± SE, unless indicated otherwise. Each n is equal to the number of animals studied, unless indicated. Only one vessel per animal was used for a given protocol.
Because concentration-response curves did not show a sigmoidal response, EC50 values were not calculated. Differences in responses between groups of arteries were determined by comparing the whole concentration-response curves by use of a two-way repeated-measures ANOVA, using substance concentration as a within-subject factor and genotype as a between-subject factor. The interaction effect between concentration and group membership tested the hypothesis that the concentration-response curves differ between the groups, and P < 0.05 was considered statistically significant. Baseline characteristics, initial artery diameters, contractile responses to U-46619, and maximum values were analyzed using ANOVA or Student's t-test as appropriate.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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There was no difference in body weight (in g) between WT (n = 13) and -ERKO (n = 13) females or between WT (n = 12) and -ERKO (n = 12) males (23.1 ± 0.6 vs. 25.6 ± 1.4 and 31.1 ± 1.6 vs. 33.1 ± 1.2, respectively). As expected WT and -ERKO males were significantly heavier than WT and -ERKO females (P < 0.05). There was no difference in femoral artery diameter in any of the groups studied with mean diameters (µm) of 265 ± 6 in WT females (n = 13), 250 ± 7 in -ERKO females (n = 13), 257 ± 6 in WT males (n = 12), and 269 ± 6 in -ERKO males (n = 12).
Concentration-response Curves to 17-E2, PPT, and DPN
Contractile responses to U-46619 were similar in arteries from all groups studied (data not shown). 17-E2 and PPT caused rapid vasorelaxation (within 5 min) in a concentration-dependent manner in arterial segments from -ERKO and WT mice. There was no significant difference in the concentration-response curves to 17-E2, PPT, and DPN (obtained only in arteries from WT mice) between groups or between sexes (Fig. 2, A–D), although there was a tendency for an enhanced dilatation to 17-E2 in arteries from -ERKO females compared with WT [%maximum relaxation: 31 ± 6 (n = 12) vs. 21 ± 6 (n = 12), P > 0.05]. Preliminary studies with DPN confirmed an absence of dilatory response in arteries from -ERKO mice except for minimal relaxation at the highest concentration (10 µM). There was also a tendency for greater dilatation to PPT than to 17-E2 in WT animals, but this did not achieve significance [in WT male %maximum relaxation to PPT 41 ± 8 (n = 8) vs. 28 ± 9% maximum relaxation to 17-E2 (n = 10); in WT female 31 ± 6% maximum dilation to PPT (n = 10) vs. 24 ± 5% maximum relaxation to 17-E2 (n = 12) P > 0.05; Fig. 2, B and D].
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-E2, PPT, and DPN Before and After NOS Inhibition
Female arteries.
NOS inhibition was associated with a reduction in 17-E2-induced dilatation across the concentration-response curve (P < 0.05) in arteries from -ERKO females, together with a significant reduction in the maximal response [%maximum relaxation: 31 ± 6 before vs. 12 ± 4 after L-NAME (n = 11), P < 0.05: Fig. 3A]. In contrast, relaxation was not affected by NOS inhibition in WT females (Fig. 3B). NOS inhibition significantly (P < 0.05) reduced dilatation to PPT across the concentration response in arteries from both -ERKO and WT females, and there was a significant reduction in the maximum response [-ERKO %maximum relaxation: 36 ± 8 before vs. 17 ± 5 after L-NAME (n = 12), P < 0.05 (Fig. 4A); in WT, %maximum relaxation: 32 ± 6 before vs. 14 ± 5 after L-NAME (n = 8) P < 0.05; Fig. 4B].
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-ERKO males, as in females, NOS inhibition led to a significant reduction in relaxation to 17-E2 across the concentration-response curve and resulted in a significant decrease in the maximum response [%maximum relaxation: 26 ± 6 before vs. 14 ± 4 after L-NAME (n = 9), P < 0.05; Fig. 6A]. In common with the response in females arteries, there was no effect of NOS inhibition on 17-E2-mediated relaxation in arteries from WT males either across the concentration-response curve or in the maximum response (n = 8; Fig. 6B).
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-ERKO females, NOS inhibition did not influence either the concentration-response curve to PPT nor the maximum response (n = 10, P > 0.05; Fig. 7A) but significantly decreased vasodilatation in arteries from WT mice across the concentration-response curve and reduced the maximum response [%maximum relaxation: 41 ± 7 before vs. 16 ± 3 after L-NAME (n = 7), P < 0.05; Fig. 7B].
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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may exert a hitherto unrecognized inhibitory influence on the acute dilatory response to 17-E2 in WT mice and that this is achieved through downregulation of ER--mediated NO release. The finding is based on the observations that, in isolated small femoral arteries from the -ERKO mouse, the NO pathway played an important role in relaxation to 17-E2 and to the selective ER- agonist PPT, whereas in the WT mouse with intact ER- and ER-, dilatation to 17-E2 was NO independent. In WT mice, the lack of an NO contribution to acute dilatation induced by DPN (ER- agonist), in contrast to an obvious role in relaxation to PPT (ER- agonist), also strengthens the suggestion of interplay between ER subtypes. The absence of ER-, however, did not affect the magnitude of dilatory responses to either 17-E2 or a selective ER- agonist (PPT).
The acute estrogen-mediated NO contribution observed in arteries from -ERKO but not in WT mice might be explained by ER- acting as a transdominant repressor on ER- or by ER- acting to modulate ER- biological activity (41). An antagonistic "Ying-Yang" relationship between these receptor subtypes has been reported previously in isolated cell lines, bone, and liver (26, 31), and we are the first to show that such interrelationship occurs in small femoral vasculature of the WT mice. Because the prevalence of each ER subtype varies with gender and the type of vascular bed (3, 6, 9, 19), the relative expression and distribution of the two ER isoforms could also predetermine the acute cellular responses to 17-E2.
In accordance with our observations in WT animals, previous investigations, which have used similar concentrations of 17-E2 to those used in the present investigation (10 nM-10 µM), also failed to demonstrate an obvious role for NO in the acute vasodilator effect to 17-E2. These include studies on isolated arteries from rats (aorta, coronary, and mesenteric arteries; see Refs. 1, 36, 39, 48), rabbit carotid artery (46), mouse aorta (13), coronary microvessels from female or male dogs (24), and human vessels, including coronary arteries (11) and from our laboratory small subcutaneous arteries from men (unpublished data). In contrast, others have implied that NO plays a role in the acute dilation to 17-E2 in, for example, isolated female coronary arteries (4, 11), aorta and femoral arteries from rat (5, 51), human mammary artery (38), rabbit coronary artery, and uterine arteries from sheep (44, 55). The lack of conformity between studies reporting the acute actions of 17-E2 remains unexplained, but in light of our observations it might be attributed to the ERs interplay and relative differences in ER- and ER- expression in the different vascular beds studied.
In our study, it is presumed that ER activation of NO-induced dilation is via activation of eNOS. Endothelial function was confirmed by substantial relaxation to ACh; hence, it is unlikely that the NO contribution to acute dilatory responses after application of estrogenic compounds was because of activation of inducible and/or neuronal NOS (15, 45).
The selective stimulation of ER- with PPT led to NO-mediated dilatation in the femoral arteries from female and male WT mice and in -ERKO female mice. We have also observed similar responses in isolated small subcutaneous arteries from healthy men (unpublished data). The mechanisms of acute NO pathway activation subsequent to ER activation are increasingly understood. ER-, colocalized within the caveolae of the plasma membrane, has been shown to mediate the acute activation of eNOS by activation of the mitogen-activated protein kinase and phosphatidylinositol 3-kinase-Akt kinase pathways (6, 12, 16, 49) through the recently identified ER--binding protein striatin (29).
It is not clear why there was no obvious NO component in PPT-mediated relaxation in arteries from -ERKO male mice. Although the reduced dilatation to 17-E2 upon NOS inhibition in -ERKO males strengthens the case for a central role of ER- in NO release, the absence of an NO component in PPT relaxation may imply that the disruption of ER- in males also modifies the activity or expression of ER-. However, in preliminary semiquantitative assessment by immunohistochemistry, we found a similar distribution of ER- in the endothelium and in the vascular smooth muscle in arteries from animals of both genders. Additionally, it should be noted that the responses to PPT after NOS inhibition showed a considerable intervariability, and a type II error cannot be discounted.
Although we have suggested that ER- may reduce NO-mediated dilatation, others have suggested that ER- may also activate eNOS but through different pathways (50). In our study, vasodilation in the femoral artery to the ER- agonist DPN did not show a significant NO component in the WT mice, which substantiates our hypothesis that this receptor tonically downregulates ER-- and NO-mediated dilatation in this vascular bed. The decreased dilatation at the highest concentration (10 µM) of DPN in the male mice after incubation with L-NAME may indicate that at this concentration DPN acts as a nonspecific ER agonist (2), although DPN is 70 times more selective for ER- compared with ER- (33). In the healthy human forearm vasculature in vivo, another partial ER- agonist (genistein; see Ref. 23) evokes acute vasodilatation via NO (56), also substantiated from ex vivo studies in rodents (e.g., rat aorta and pulmonary arteries; see Refs. 10 and 34). Genistein has, however, some affinity for ER- (23) and has been shown to inhibit tyrosine kinase (20) and to activate eNOS via protein kinase A in an ER-independent manner (22, 27).
If, as suggested, ER- downregulates ER--mediated NO contribution in the mouse femoral artery, it would be anticipated in the WT mouse that relaxation to 17-E2 would be much reduced compared with that in the -ERKO arteries, whereas we found it to be insignificantly affected. This implies that ER-/ER- interplay in the WT mouse leads to dilatation through NO-independent pathways, which, as reported previously, might be the result of a direct action of the estrogenic compounds on calcium and ion channels dynamics (37, 43), cAMP cascade (27), contribution of prostacyclin (14), and endothelium-derived hyperpolarizing factor (47). These pathways could also be relevant for the residual relaxation observed in our study after NOS inhibition.
In contrast to an investigation of rat isolated small mesenteric arteries, in which PPT evoked a significantly larger vasodilator effect and DPN a significantly smaller effect compared with 17-E2 (35), we found no difference in dilatory responses to these agonists between strains or between males and females. Again, discrepancies between this study and the previous report may reflect either species or tissue differences (2).
In summary, acute dilatory responses induced by different estrogenic compounds in small femoral arteries are similar in WT and -ERKO mice of both genders but differ mechanistically. The presence of ER- apparently inhibits ER--mediated NO contribution, suggesting interaction between ERs and supporting the importance of ER subtype interplay in the maintenance of endothelial function.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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M. N. Cruz and G. Douglas contributed equally to this study.
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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REFERENCES |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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  T. Lahm, P. R. Crisostomo, T. A. Markel, M. Wang, Y. Wang, J. Tan, and D. R. Meldrum Selective estrogen receptor-{alpha} and estrogen receptor-{beta} agonists rapidly decrease pulmonary artery vasoconstriction by a nitric oxide-dependent mechanism Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2008; 295(5): R1486 - R1493. [Abstract] [Full Text] [PDF]
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 M. N. Cruz, L. Luksha, H. Logman, L. Poston, S. Agewall, and K. Kublickiene Acute responses to phytoestrogens in small arteries from men with coronary heart disease Am J Physiol Heart Circ Physiol, May 1, 2006; 290(5): H1969 - H1975. [Abstract] [Full Text] [PDF]
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