Medroxyprogesterone acetate does not antagonize estrogen-induced increases in endothelium-dependent vasodilation: potential clinical implications

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1 FERTILITY AND STERILITY VOL. 78, NO. 1, JULY 2002 Copyright 2002 American Society for Reproductive Medicine Published by Elsevier Science Inc. Printed on acid-free paper in U.S.A. Medroxyprogesterone acetate does not antagonize estrogen-induced increases in endothelium-dependent vasodilation: potential clinical implications Hillary Dinh, B.S., a and Lauren Nathan, M.D. b David Geffen School of Medicine at University of California, Los Angeles, California Received January 3, 2002; revised and accepted March 1, Supported in part by a predoctoral fellowship from the American Heart Association, Southern California chapter (H.D.) and by a Pfizer/Society for Women s Health Research Scholars Grant for Faculty Development in Women s Health (L.N.). Also supported in part by National Institute of Aging grant AG Reprint requests: Lauren Nathan, M.D., UCLA School of Medicine, , CHS, Los Angeles, California (FAX: ; lnathan@mednet. ucla.edu). a Department of Molecular and Medical Pharmacology. b Department of Obstetrics and Gynecology /02/$22.00 PII S (02) Objective: Determine the effect of combined 17 -estradiol benzoate (E 2 ) and medroxyprogesterone acetate (MPA) administration on endothelium-dependent vasorelaxation to acetylcholine (ACh). Design: Prospective, ex vivo study. Setting: Academic research laboratory. Animal(s): Mature female rats. Intervention(s): Ovariectomized rats received one of the following interventions daily for 3 days: [1] corn oil via IM injection, [2] E 2 (20 g/kg IM), or [3] E 2 (20 g/kg IM) and MPA (10 mg/kg IM). Main Outcome Measure(s): Basal release of nitric oxide (NO) and endothelium-dependent and endothelium-independent vasodilation from thoracic aortas obtained from each group. Result(s): Estradiol treatment potentiated the endothelium-dependent relaxation to ACh when compared with the control group. Administration of MPA with E 2 did not antagonize the beneficial effect of E 2 on endothelium-dependent relaxation. Estradiol treatment alone or in combination with MPA did not affect endothelium-independent vasodilation as compared with the case of the control group. Administration of E 2 resulted in increased basal NO release (assessed indirectly by measuring the constrictor response to N G -nitro- L-arginine [methyl ester (L-NAME)]) when compared with the case of the control group, and the addition of MPA to E 2 did not alter the effect of E 2 on basal NO release. Conclusion(s): Estradiol potentiates endothelium-dependent relaxant responses and increases basal endothelial NO release. Medroxyprogesterone acetate does not antagonize these effects of E 2. (Fertil Steril 2002;78: by American Society for Reproductive Medicine.) Key Words: Estradiol, medroxyprogesterone acetate, nitric oxide, L-NAME, endothelium-dependent vasodilation Estrogen therapy has been shown to have a cardioprotective effect in postmenopausal women (1, 2), and many mechanisms may be involved. Endothelial dysfunction characterized by loss of endothelium-dependent vasodilation is an early physiologic event in atherogenesis (3). Several studies reported that estrogen prevents endothelial dysfunction (4, 5) by increasing the production and release of nitric oxide (NO) from the endothelium (6 8). Nitric oxide is a vasodilator (9 11) and prevents adhesion of platelets to endothelial cells (12). Nitric oxide also suppresses the expression of the vascular cell adhesion molecule (VCAM-1) (13, 14) and of monocyte chemoattractant protein-1 (MCP-1) (15). The attenuation of monocyte adhesion and transendothelial migration after estrogen administration observed in rabbits (16) may therefore occur via an NO-mediated mechanism. Nitric oxide can be released from the endothelial cells by various endothelium-dependent vasodilators like acetylcholine (ACh) (9) and bradykinin (9), as well as after increases in vascular tone (tonerelated release) (17). Despite the beneficial effects of estrogens on the coronary vasculature, estrogens induce endometrial hyperplasia and increase the risk of endometrial cancer in postmenopausal women (18). For these reasons, estrogen therapy must be given along with a progestational agent in women with a uterus to prevent hy- 122

2 perplasia and cancer of the endometrium (19 21). Medroxyprogesterone acetate, one of the most widely used progestins in clinical practice, was reported to antagonize the reduction of atherosclerotic plaque size in monkeys that were administered estrogen (22). In addition, MPA abolished the estrogen-induced increases in endothelium-dependent vasodilation produced by ACh in atherosclerotic monkeys (23). Questions have therefore been raised as to whether medroxyprogesterone acetate should be used as the progestational agent for women on HRT. The purpose of our study was to assess the effects of MPA on estrogen-mediated changes in endothelium-dependent vasodilation and NO release in rat aortic rings to elucidate whether the effect of MPA in antagonizing the beneficial effects of estrogen on endothelial cells observed in the subhuman primate model (23) was specific to this species or whether it could be applicable across species. MATERIALS AND METHODS Drugs and Materials Phenylephrine hydrochloride, ACh perchlorate, glyceryl trinitrate (GTN), N G -nitro-l-arginine methyl ester (L- NAME) hydrochloride, and 17 -estradiol benzoate (E 2 ) were purchased from Sigma Chemical (St. Louis, MO); and 150 mg/ml MPA (Depo-Provera) injectable suspension was purchased from Pharmacia & Upjohn (Kalamazoo, MI). Estradiol was dissolved in corn oil to the final concentration of 20 g/ml. Medroxyprogesterone acetate (150 mg/ ml) was diluted with saline to the final concentration of 10 mg/ml on the day of injection. Animals Female Sprague-Dawley rats weighing between 175 g and 200 g were purchased from Charles River Laboratories (Boston, MA). Ovariectomy was performed under anesthesia (ketamine, 100 mg/kg and xylazine, 10 mg/kg; IM). The ovaries were ligated and removed. Animals were then allowed to recover for 3 days with food and water ad libitum. Three days after oophorectomy, the rats were randomly divided into three groups, and hormone therapy was initiated for 3 days. The first treatment group (n 10) received vehicle (200 L of corn oil IM daily). The second treatment group (n 10) received daily injection of E 2 (20 g/kg in 200 L corn oil, IM); this dose was chosen based on the fact that 20 g/kg of 17 -estradiol protected against neointima injury in the balloon-injured rat model (24). The third group (n 10) received E 2 (20 g/kg) and MPA (10 mg/kg IM in 200 L of injection volume) given daily as separate injections; this dose was chosen based on the work of other investigators who administered 10 mg/kg of MPA to rats and found this dose to be effective in attenuating the antiproliferative effects of estrogen in the rat carotid artery injury model (25). All procedures were done in accordance with current National Institutes of Health guidelines and were approved by the UCLA Animal Research Committee. Preparation of Rat Aortic Rings and Tension Recording The preparation of rat aortic rings was similar to that described by Furchgott and Zawadzki (9). Briefly, the rats were sacrificed by an overdose of pentobarbital (50 mg/kg intraperitoneally). The thoracic aortas were carefully removed to protect the endothelial lining, cleared of adhering fat and connective tissues, and cut transversely into segments 5 mm in length. The rings were mounted under1gofresting tension on stainless-steel hooks in 25-mL capacity muscle chambers and bathed in Krebs bicarbonate solution, ph 7.4 at 37 C (composition: 122 mm NaCl, 4.76 mm KCl, 25 mm NaHCO 3, 1.18 mm NaH 2 PO 4 H 2 O, 1.25 mm CaCl 2, 1.18 mm MgSO 4 H 2 O, 5.5 mm glucose) (26). Tension was measured isometrically using force displacement (FT 03C) transducers and displayed either on a Grass polygraph (model 79D) or on a Soltec (model 1243) recorder. After the rings were allowed to equilibrate to 1 g tension, Krebs solution was replaced with 122 mm KCl solution to determine the maximal contractile response of the aortic rings. After the maximal response induced by high KCl, the rings were washed with Krebs solution to equilibrate back to resting tension. Tissue that did not constrict by 0.5 g was not used. The viability and functional integrity of the endothelium in each aortic ring were initially determined by contracting the rings with phenylephrine ( M) and then observing the relaxation to 10 6 Mof ACh. In experiments in which we used endothelium-denuded aortic rings, the absence of a response to ACh was observed, confirming that the aortic rings were denuded of endothelium. Experiment 1. Effect of E 2 and MPA on Endothelium-Dependent Vasodilation Induced by ACh Experiments were performed to elucidate the effects of administration of either E 2 alone or E 2 in combination with MPA on the responsiveness of endothelium-intact aortic rings to an endothelium-dependent vasodilator such as ACh. In these experiments, submaximal tension (70% 80% of the contractile response to 122 mm KCl) was initially induced with phenylephrine (10 6 M), after which cumulative concentration relaxant responses were obtained with ACh ( M). The relaxation response to ACh was calculated as the percentage of decrease in tension below the evaluated tension elicited by precontracting arterial rings with phenylephrine. Experiment 2. Effect of E 2 and MPA on the Basal (Tone-Related) Release of NO In this experiment, concentration-related contractile responses to L-NAME (0.01 M to100 M), an inhibitor of FERTILITY & STERILITY 123

3 NO synthesis, were assessed (27). To elucidate the basal (tone-related) release of NO from endothelium-intact aortic rings, moderate vascular tone (35% 50% of the contractile response to 122 mm KCl) was induced with low concentrations of phenylephrine ( M). When the response to phenylephrine equilibrated, cumulative concentrations of L-NAME were added every 15 minutes, starting with the lowest concentration of L-NAME. Contraction responses to different concentrations of L-NAME were calculated as the percentage of increase in tension above the induced tension that was elicited by precontracting arterial rings with phenylephrine. Experiment 3. Effect of E 2 and MPA on Endothelium-Independent Vasodilation Produced by GTN Endothelium-denuded aortic rings were used to determine the cumulative endothelium-independent relaxant responses to GTN ( M), an endothelium-independent vasodilator. Aortic rings were initially preconstricted with phenylephrine to submaximal tension (70% 80% of the contractile response to 122 mm KCl). The relaxation response to cumulative concentrations of GTN was calculated as the percentage of decrease in tension below the initially induced tension elicited by precontracting arterial rings with phenylephrine. Experiment 4. Effect of E 2 and MPA on the Uterine Weight To assess the efficacy of the steroids administered, uterine horns from individual rats in each group were collected and weighed. Statistical Analysis All data are expressed as mean SEM. For nonlinear regression of the data fit to the sigmoidal dose response curve, GraphPad Prism software (San Diego, CA) was used. EC 50 s were determined from the best-fit values for log effective concentration (EC) 50 s. The F test was used to compare the differences between the dose response curves (28). The data for uterine weight were analyzed by one-way ANOVA followed by post hoc Tukey test to determine the differences between the groups. The differences were considered statistically significant at P.05. All data were analyzed by GraphPad Prism software. RESULTS Effect of E 2 and MPA on Endothelium- Dependent Vasodilation Induced by ACh Acetylcholine produced concentration-dependent relaxation of phenylephrine-precontracted aortic rings (Fig. 1). Administration of E 2 either alone (EC M) or in combination with MPA (EC M) significantly enhanced the relaxant response to ACh and shifted the dose response curve to the left as compared with the control group (EC M, P.001). There was FIGURE 1 Effects of E 2 and MPA on endothelium-dependent vasodilation produced by ACh. Mean SEM percentage relaxation of endothelium-intact aortic rings from ovariectomized (OVX) female rats in response to different concentrations of ACh. Aortic rings were submaximally contracted (70% 80% of that produced by 122 mm KCl) with phenylephrine before obtaining cumulative responses to ACh. Relaxation was significantly enhanced in aortic rings from E 2 ( )-treated or E 2 plus MPA (Œ) treated OVX rats as compared with the placebo-treated group ( ). * P.001 compared with placebo group. The concentration response curves from E 2 -treated or E 2 plus MPA treated rats overlapped and showed no statistically significant difference between the two groups (P.05). no statistically significant difference between E 2 -treated and E 2 plus MPA treated groups (P.05; Fig. 1). All three experimental groups reached the same maximal response (Fig. 1). Effect of E 2 and MPA on the Basal (Tone- Related) Release of NO L-NAME methyl ester elicited vasoconstriction in a concentration-dependent and endothelium-dependent manner (Fig. 2). The maximal vasoconstriction produced by L-NAME was significantly greater in aortic rings from the E 2 -treated group (192% contraction) and the E 2 plus MPA treated group (181% contraction) when compared with the control group (59.5% contraction; P.001). There was no statistically significant difference between the E 2 alone treated and E 2 plus MPA treated groups (P.05). Administration of E 2 alone or E 2 in combination with MPA caused the concentration response curves to L-NAME to be significantly shifted to the left (Fig. 2) as compared with the control group (P.001). There was no significant difference between the E 2 alone treated and E 2 plus MPA treated groups (P.05). 124 Dinh and Nathan Medroxyprogesterone acetate and vasomotion Vol. 78, No. 1, July 2002

4 FIGURE 2 Effects of E 2 and MPA on the tone-dependent release of NO. Mean SEM percentage contraction of endothelium-intact aortic rings from ovariectomized (OVX) female rats in response to different concentrations of N G -nitro-l-arginine methyl ester (L-NAME). The aortic rings were moderately contracted (30% 50% of that produced by 122 mm KCl) by phenylephrine before cumulative responses were obtained to L-NAME. The magnitude of contraction was significantly greater in aortic rings from E 2 )( )-treated or E 2 plus MPA (Œ) treated OVX rats. * P.001 compared with placebo group ( ). There was no significant difference between E 2 or E 2 plus MPA treated rats (P.05). FIGURE 3 Effect of E 2 and MPA on endothelium-independent vasodilation induced by GTN: mean SEM percentage relaxation of endothelium-denuded aortic rings from ovariectomized (OVX) female rats in response to different concentrations of GTN. Aortic rings were submaximally contracted (70% 80% of contraction produced by 122 mm KCl) with phenylephrine before cumulative responses to GTN were obtained; responses were similar in all three groups (P.05)., placebo;, E 2 ; Œ, E 2 plus MPA. Effect of E 2 and MPA on the Endothelium- Independent Vasodilation Produced by GTN Phenylepherine-preconstricted, endothelium-denuded aortic rings relaxed in response to GTN in a concentration-dependent manner (Fig. 3). Administration of E 2 alone (EC M) or in combination with MPA (EC M) did not alter the concentration-dependent relaxant response to GTN (Fig. 3) when compared with the control rings (EC M). No significant differences in the relaxant responses to GTN were seen in aortic rings from all three experimental groups (P.05). Effect of E 2 and MPA on the Uterine Weight In animals that were treated with E 2, uterine weight significantly increased (0.50 g) when compared with that of those obtained from control animals (0.25 g, P.001) (Fig. 4). When animals were treated with a combination of E 2 and MPA, the uterine weight was significantly less (0.36 g) when compared with the E 2 alone treated group and was comparable to that of the control group (P.05) (Fig. 4). The role of progestational agents, especially MPA, in modulating the beneficial effects of estradiol on the endothelium-dependent relaxation to ACh is controversial. In studies in subhuman primates, the increase in ACh-induced vasodilation of coronary vessels after administration of estradiol to ovariectomized monkeys was antagonized by MPA FIGURE 4 Effect of E 2 and MPA on uterine weight. Mean SEM uterine weight. Uterine horns from individual rats in each group were collected and weighed. Uterine weight was significantly higher from E 2 -treated rats when compared with placebotreated or E 2 plus MPA treated rats. * P.001 compared with placebo group. P.001 compared with E 2 -treated rats. DISCUSSION FERTILITY & STERILITY 125

5 (23). The primary objective of this study, therefore, was to assess whether the adverse effects of MPA on endothelial function were just species specific, restricted to subhuman primates, or could be generalized to other species. We therefore elected to assess the effects of E 2 administration, either alone or in combination with MPA, on ACh-induced endothelium-dependent relaxation as well as basal release of NO. We selected the rat aortic ring as our model system because it has been used by numerous investigators to study endothelium-dependent dilation and release of NO (9). Moreover, estrogens modulate ACh-induced endotheliumdependent vasodilation, as has been observed in subhuman primates (29) and women (30, 31). Acetylcholine was selected because it consistently produces endothelium-dependent relaxation of rat aortic rings by a receptor-mediated mechanism. Acetylcholine is a potent releaser of NO from endothelial cells of the conduit vessels (32). Our finding that endothelium-dependent vasodilation to ACh was increased in aortic rings obtained from E 2 -treated animals when compared with those obtained from ovariectomized animals suggests that estrogens may have increased the number and/or efficacy of the muscarinic receptors to release NO. The basal (tone-dependent) release of NO was assessed indirectly by initially inducing moderate tone and then observing the effects of L-NAME on changes in basal tone. L-Arginine is converted to L-citrulline in endothelial cells (33, 34) by the enzyme NO synthase (35), and NO is formed as a byproduct of this reaction. Nitric oxide synthesis can therefore be inhibited by certain analogues of L-arginine (36, 37), such as L-NAME. Differences in basal NO formation would therefore be reflected in the amount of contraction of the endothelium-intact aortic rings in the presence of arginine analogs. After the addition of L-NAME to the muscle bath, the contraction of endothelium-intact aortic rings from ovariectomized rats treated with estradiol was significantly greater in magnitude than that of aortic rings from ovariectomized rats not treated with hormones. The contractile effects of this arginine analog were not seen in the presence of threefold to fourfold excess of L-arginine, indicating that the effects seen were, indeed, due to increased NO synthesis from L-arginine. Our observation that aortic rings from estradiol-treated animals had increased basal release of NO when compared with those obtained from ovariectomized rats is similar to that observed previously in rabbits (6) and appears to be due to increased NO synthase, as has been shown by numerous investigators (38). The absence of any differences in the responsiveness of aortic rings to GTN indicates that the observed differences in the basal and ACh-induced release of NO between aortic rings obtained from ovariectomized and estradiol-treated animals and ovariectomized animals not receiving hormones is solely due to an increased release of NO from endothelial cells and is not due to increased sensitivity of the vascular smooth muscle to the NO released. Our observation that MPA did not antagonize the estradiol-induced increase in the ACh response as well as the basal release of NO (despite its antagonizing the estradiolinduced increase in uterine weight) indicates that the effects of MPA reported in subhuman primates in antagonizing the beneficial effects of E 2 on the endothelium are most likely species specific. This interpretation is supported by the observation that chronic coadministration of a progestational agent, including MPA, in postmenopausal women without risk factors for coronary artery disease did not appear to adversely affect the beneficial effect of estrogen on endothelial function (39), unlike that seen in subhuman primates (23). One plausible explanation for the differences in the action of MPA observed in subhuman primates compared with that observed in rats and women may be potentially due to some metabolite of MPA specific to subhuman primates that is not produced either in rats or in women and which may adversely affect endothelial function. Medroxyprogesterone acetate has structural similarities to progesterone, and progesterone is metabolized differently in subhuman primates when compared with the case in humans (40, 41). Another interesting feature observed in our study is that MPA was very effective in antagonizing the effects of estradiol on the uterus but not of that in endothelial cells. Further studies need to be carried out to elucidate the mechanism underlying this paradox. Further studies also need to be carried out to assess other potential mechanisms that may explain why MPA antagonizes the endothelium-dependent and basal release of NO in subhuman primates, an effect not observed in rats or in women. References 1. Stampfer MJ, Colditz GA. Estrogen replacement therapy and coronary heart disease: a quantitative assessment of the epidemiologic evidence. Prev Med 1991;20: Green A, Bain C. Epidemiological overview of estrogen replacement and cardiovascular disease. Baillieres Clin Endocrinol Metab 1993;7: Vita JA, Treasure CB, Nabel EG, McLenachan JM, Fish RD, Yeung AC, et al. Coronary vasomotor response to acetylcholine relates to risk factors for coronary artery disease. 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