The Pathway-Selective Estrogen Receptor Ligand WAY-169916 Reduces Infarct Size After Myocardial Ischemia and Reperfusion by an Estrogen Receptor Dependent Mechanism

Abstract

Previous studies have shown that estrogen treatment protects the heart from reperfusion injury. The adverse effects of long-term estrogen treatment limit its clinical use and emphasize the need for the development of specific pharmacological interventions such as pathway-selective estrogen receptor (ER) ligands. Pathway-selective ER ligands are compounds that retain estrogen's anti-inflammatory ability, but they are devoid of conventional estrogenic action. In the present study, the pathway-selective ER ligand WAY-169916 was assessed for its cardioprotective potential in an in vivo model of ischemia-reperfusion injury. Anesthetized, ovariectomized rabbits were administered WAY-169916 (1 mg/kg), 17β-estradiol (E2; 20 μg/rabbit), or vehicle intravenously 30 minutes before a 30-minute occlusion and 4 hours of reperfusion. Acute treatment with either WAY-169916 or E2 resulted in a decrease in infarct size, expressed as a percent of area at risk (WAY-169916, 21.2 ± 3.3; P < 0.001 and E2, 18.8 ± 1.7; P < 0.001) compared with vehicle 59.4 ± 5.4). Pretreatment with estrogen receptor antagonist ICI 182,780 significantly limited the infarct size sparing effect of both WAY-169916 and E2 when expressed as a percent of the risk region (WAY 169916, 47.4 ± 4.4; E2, 53.01 ± 5.0). The results demonstrate that WAY-169916 protects the heart against ischemia-reperfusion injury through an ER-dependent mechanism.

INTRODUCTION

Estrogens are a family of structurally related hormones that play a role in many tissue types affecting physiological functions in both men and women. In addition to the classical reproductive target tissues, there is estrogenic action in non-classic tissues, including brain, bone, cardiovascular system, immune system, and liver.^1-3 Estrogen deficiency seems to be involved in many pathological processes such as arteriosclerosis, osteoporosis, and degenerative processes in the CNS, as well as the increased risk of cardiovascular disease after menopause.^4-6

A potential role for estrogen in cardiovascular protection has been suggested by the observation that women have a reduced risk of cardiovascular disease compared with men, and this benefit is lost after menopause when estrogen levels drastically decrease. Experimental evidence suggests that biologically active 17β-estradiol (E2) reduces both the extent of irreversible myocardial injury as well as the incidence and duration of reperfusion-induced ventricular tachycardia and ventricular fibrillation.⁷ Myocardial infarct size resulting from coronary artery occlusion and reperfusion was reduced in male rabbits after acute treatment with E2 whereas 17α-estradiol, the inactive form of the hormone, showed no benefit.⁸ The importance of the estrogen receptor (ER) in cardioprotection was further implicated when the infarct size-limiting effect of E2 was inhibited by the ER antagonist ICI 182,780.⁹ In addition to estrogen's ability to induce vasorelaxation¹⁰ and act as a free radical scavenger^11,12 and an antioxidant,¹³ estradiol bound to either ERα or ERβ has been shown to inhibit nuclear factor-κB (NF-κB) transcriptional activity.¹⁴ Each of these actions may have a role in the reduction of myocardial infarct size; however, the exact mechanism remains to be defined.

The search for more acceptable and safer postmenopausal hormonal therapies has led to the evaluation of compounds known as pathway-selective ER ligands.¹⁵ Pathway-selective ER ligands are compounds that retain anti-inflammatory effects of estrogen, but they are devoid of conventional estrogenic action. The pathway-selective ER ligand WAY-169916 was designed to retain ER-dependent antagonism of inflammatory signaling cascades while having no activity on classical ER-mediated transcription.¹⁵ The present study was designed to determine the effects of acute administration of WAY-169916 on infarct size. We hypothesized that WAY-169916 would reduce infarct size and the inflammatory response to ischemia and reperfusion. We further designed studies to determine if the protective effects of WAY-169916 are mediated by a receptor-dependent action.

METHODS

Guidelines for Animal Research

The procedures used in this study are in agreement with the guidelines of the University of Michigan Committee on the Use and Care of Animals. The University of Michigan Unit for Laboratory Animal Medicine provides veterinary care. The University of Michigan is accredited by the American Association of Accreditation of Laboratory Animal Health Care, and the animal care use program conforms to the standards in “the Guide for the Care and Use of Laboratory Animals,” DHEW Publ. N. (NIH) 86-23.

Surgical Preparation

Six to eight weeks after ovariectomy, New Zealand white rabbits (2.8 to 3.2 kg) were anesthetized with a combination of xylazine (3.0 mg/kg) and ketamine (35 mg/kg) administered intramuscularly and followed by an intravenous injection of sodium pentobarbital (15 mg/kg). After insertion of a cuffed endotracheal tube, the animals were placed on positive-pressure ventilation using room air. The left jugular vein was isolated and cannulated for drug administration. The left carotid artery was isolated and instrumented with a Millar catheter micro-tip pressure transducer (Millar Inst. Inc, Huston, TX) positioned immediately above the aortic valve to monitor aortic blood pressure. The lead II electrocardiogram was monitored throughout the protocol. A left thoracotomy and pericardiotomy were performed, followed by identification of the left anterior descending coronary artery. A silk suture (3-0; Deknatel, Fall River, MA) was passed under the artery and around a short length of polyethylene tubing. Simultaneous downward displacement of the polyethylene tubing while applying upward traction on the suture resulted in occlusion of the coronary artery and cessation of regional myocardial blood flow. Coronary artery occlusion was maintained for 30 minutes, after which time reperfusion was initiated by withdrawing the polyethylene tubing. Regional myocardial ischemia was verified by the presence of a zone of cyanosis in the area of distribution of the occluded vessel and by changes in the electrocardiogram consistent with the presence of transmural regional myocardial ischemia (ST - segment elevation).

Experimental Protocol

The animals were allowed to stabilize for 15 minutes before beginning the protocol, which involved 2 experimental groups. Group 1 consisted of 42 rabbits randomized equally among 3 treatments: WAY-169916 (1 mg/kg; n = 14), E2 (20 μg/rabbit; n = 14), or vehicle (1 mL 20% DMSO 80% PEG; n = 14) administered 30 minutes before occlusion of the left anterior descending coronary artery. Rabbits in Group 2 were treated with the estrogen receptor antagonist ICI 182,780 (1 mg) for 1 hour before acute treatment with the pathway-selective ER ligand WAY-169916 (n = 14), E2 (n = 14), or vehicle (20% DMSO 80% PEG; n = 14).

Determination of Infarct Size

Hearts from 5 animals per group were used to determine infarct size. At the completion of the 4-hour reperfusion period, the hearts were removed, the aorta was cannulated, and the coronary vascular bed was perfused on a Langendorff apparatus with Krebs-Henseleit Buffer at a constant flow of 22-24 mL/min. The hearts were perfused with buffer for 10 minutes to clear the vascular compartment of plasma and blood cellular elements. Forty-five milliliters of a 1% solution of 2,3,5-triphenyltetrazolium chloride (TTC) in phosphate buffer (pH 7.4, 37°C) was perfused through the coronary vascular bed. TTC demarcates the non-infarcted myocardium within the area at risk with a brick-red color, indicating the presence of a formazan precipitate resulting from the reduction of TTC by dehydrogenases present in viable myocardial tissue. Irreversibly injured tissue, lacking cytosolic dehydrogenases, is unable to form the formazan precipitate and appears pale yellow. Upon completion of the TTC infusion, the left circumflex coronary artery was ligated at the site identical to that ligated during the induction of regional myocardial ischemia. The perfusion pump was stopped, and 3 mL of a 0.25% solution of Evans Blue was injected slowly through a side-arm port connected to the aortic cannula. The dye was passed through the heart for 10 seconds to ensure its uniform tissue distribution. The presence of Evans Blue was used to demarcate the left ventricular tissue that was not subjected to regional ischemia, as opposed to the risk region. The heart was removed from the perfusion apparatus and cut into 3 transverse sections at right angles to the vertical axis. The right ventricle, apex, and atrial tissue were discarded. In an unblinded fashion, the executor of the protocol traced both surfaces of each transverse section onto clear acetate sheets. The traced sections were scanned and downloaded into Adobe PhotoShop (Adobe Systems Inc. Seattle, WA). The areas of the normal left ventricle non-risk region, area at risk, and infarct region were determined by calculating the number of pixels occupying each area using the Adobe PhotoShop software. Total area at risk is expressed as the percentage of the left ventricle. Infarct size is expressed as the percentage of the area at risk.

Evaluation of Neutrophil Accumulation

Tissue samples to be assayed for myeloperoxidase (MPO) activity were obtained from ventricular tissue sections that had not been reacted with TTC to avoid interference with the enzymatic assay (6 animals per group). Samples of area at risk (AAR) were obtained from myocardium immediately distal to the site of vessel occlusion. Samples of left ventricle and AAR were weighed and immediately frozen in liquid nitrogen until assayed. Tissue was placed in buffer (50 mM sodium phosphate, pH 6.0) and homogenized with a polytron homogenizer (Tekmar Co., Cincinnati, OH). The homogenates were centrifuged for 30 min (3000 × g, 4°C), and the supernatants were removed. MPO activity was determined by measuring the change in absorbance at 460 nm resulting from the conversion of H₂O₂ in the presence of O-dianisidine (Sigma, St. Louis, MO), as described previously.¹⁶ The MPO activity was normalized to the weight of the sample.

Morphological Changes

Tissue samples used for electron microscopy were obtained from a separate group of experiments (3 animals per group). Upon completion of the designated protocol, hearts were perfused for 3 min with 2.5% glutaraldehyde and 1% LaCl3 in 0.1 M sodium cacodylate buffer (pH 7.44). The electron-dense LaCl₃ serves as an indicator of blood vessel integrity. Tissue samples from the left ventricular myocardium were removed and cut into segments measuring approximately 1 mm on a side. The samples were fixed for an additional 2 hours at room temperature and then overnight at 4°C. After washing with 0.1 M sodium cacodylate buffer, the samples were dehydrated in an ethanol series and embedded in EM bed-812 (Electron Microscopy Sciences). Tissue blocks were sectioned with a Reichert ultramicrotome, placed onto formvar-coated copper grids, and then stained with 4% uranyl acetate. Sections were observed with a Phillips CM-100 electron microscope.

MATERIALS

The pathway-selective ER ligand WAY-169916 was synthesized and provided by Wyeth Pharmaceuticals (Collegeville, PA).¹⁷ The estrogen receptor antagonist ICI 182,780 was provided by Wyeth Research. Structures of the compounds are shown in Figure 1. All other materials were purchased from Sigma Chemical Co. (St. Louis, MO).

FIGURE 1: Chemical structures for (A) WAY-169916, (B) E2, and (C) ICI 182,780.

Statistical Analyses

The data are expressed as the mean ± SEM. Differences between control and experimental groups were determined using a one-way analysis of variance (ANOVA) for multiple groups. Post-test differences between groups were determined using Bonferoni post-test. A value of P < 0.05 was considered to be significant

RESULTS

Hemodynamic Effects

Hemodynamic variables were obtained to determine the effects of estradiol in mediating alterations in arterial blood pressure and heart rate. An immediate decrease in arterial blood pressure followed by a return to the baseline values was observed after intravenous administration of the pathway-selective ER ligand WAY-169916, E2, or the vehicle control (data not shown). The rate-pressure product (RPP), defined as mean arterial blood pressure multiplied by the heart rate divided by 100, was used as an indicator of myocardial oxygen consumption. As shown in Table 1 the RPP decreased in each of 3 groups (WAY 169916, E2, and vehicle) from equilibration to 30 minutes after treatment and then remained stable throughout the duration of the protocol with no differences between groups. As seen in Table 1, RPP was reduced similarly in all groups treated with ICI 182,780. The RPP remained stable throughout the duration of the protocol with no significant difference between groups.

TABLE 1: Effect of E2 or WAY-169916 treatment on rate pressure product [(bpm × mmHg)/100] in ovariectomized female rabbits

Electrophysiological data did not demonstrate any changes on administration of either WAY-169916 or E2. All animals exhibited ST-segment elevation during the induction of regional myocardial ischemia. The ST-segment changes resolved towards baseline upon removal of the occlusive ligature. In all groups, premature ventricular complexes were present immediately after reperfusion. No deaths, from either cardiac arrhythmias or cardiac failure, were noted in any of the groups.

Infarct Size

Acute administration of either WAY-169916 or E2 reduced infarct size as a percent of area at risk. Rabbits treated with either WAY-169916 or E2 developed significantly smaller infarcts expressed as a percent of the area at risk compared with animals treated with vehicle (Figure 2A). The size of the area at risk expressed as a percent of the total left ventricle was similar in each of the 3 groups (Figure 2B).

FIGURE 2: Effects of WAY-169916 and E2 alone or combined with the ER antagonist ICI 182,780 on myocardial infarct size. (A) Infarct size expressed as a percent of the AAR is decreased significantly in the group treated with WAY-169916 (1 mg/kg) or E2 (20 μg). (B) The risk regions expressed as percent of left ventricle did not differ between groups. (C) Rabbits pretreated with ICI 182,780 (1 mg) and then treated with either WAY-169916 (1 mg/kg) or E2 (20 μg) developed similar size infarcts expressed as a percent of the AAR compared to rabbits treated with vehicle. (D) When expressed as a percent of left ventricle, the tissue subjected to the ischemic episode was similar in all groups. Results are expressed as the mean + SEM. ***P < 0.001 versus control.

Rabbits treated with ICI 182,780 and WAY-169916 or E2 developed similar size infarcts expressed as a percent of the area at risk compared with the control rabbits treated with vehicle (Figure 2C). The size of the area at risk or ischemic region expressed as a percent of the total left ventricle was similar in each treatment group (Figure 2D).

Neutrophil Accumulation

Treatment with either WAY-169916 or E2 reduced the extent of neutrophil accumulation into the area at risk. The administration of WAY-169916 or E2 resulted in a decrease in MPO activity in the AAR of animals compared with control, indicating a decreased neutrophil accumulation within the AAR of animals treated with WAY 169916-9 or E2 (Figure 3A). The inhibition of neutrophil accumulation in the AAR of animals treated with WAY 169916-9 or E2 was attenuated by pretreatment with the estrogen receptor antagonist ICI 182,780 (Figure 3B).

FIGURE 3: Neutrophil accumulation in the area at risk (AAR) as determined by myeloperoxidase (MPO) activity in tissues from animals treated with WAY-169916, E2, or vehicle (A) and animals treated with ICI 182,780 and WAY-169916, ICI 182,780 and E2, or vehicle (B). The MPO activity in the AAR from WAY-169916 (1 mg/kg) and E2-treated (20 μg) animals was decreased as compared with vehicle-treated animals. The decrease in MPO activity seen in the WAY-169916 and E2-treated animals was eliminated with the pretreatment of the estrogen receptor antagonist ICI 182,780 (1 mg). Values are normalized to the tissue wet weight and represent the mean ± SEM. **P < 0.01 versus control.

Morphologic Changes

WAY-169916 and E2 preserved the morphology of tissue in the AAR after ischemia and reperfusion. In the hearts from vehicle-treated animals, sarcomere structural features were altered and contraction band necrosis was present. The mitochondria were markedly swollen with disrupted cristae and osmophillic inclusion bodies. In the hearts from WAY-169916-treated and E2-treated animals, the sarcomere structure was relatively normal and the mitochondria appeared intact with only minimal swelling. The lanthanum chloride was localized to the interior of the vessel, indicating that the blood vessels are intact. The virtual absence of contraction bands in hearts from WAY-169916-treated and E2-treated animals was in marked contrast with changes observed in the heart tissue specimens obtained from the vehicle-treated animals (Figure 4).

FIGURE 4: Representative electron micrographs of hearts from control animals and animals treated with WAY-169916 and E2. In the vehicle-treated hearts, sarcomere structural features are obliterated, and contraction bands are present. The mitochondria are markedly swollen with disrupted cristae and osmophillic inclusion bodies. In the WAY-169916 (1 mg/kg) and E2 (20 μg)-treated hearts, the sarcomere structure is relatively normal, the mitochondria appear intact with only minimal swelling, and LaCl₃ is restricted to the interior of the blood vessels. The virtual absence of contraction bands stands in marked contrast with those observed in the control hearts. Magnification: ×19000.

ICI 182,780 prevents the protection of tissue morphology afforded by WAY-169916 and E2. In all of the treatment groups (ie, vehicle, ICI 182,780 plus WAY-169916, and ICI 182,780 plus E2), sarcomere structural features were altered and contraction bands were present. The mitochondria were swollen with disrupted cristae and osmophillic inclusion bodies (Figure 5).

FIGURE 5: Representative electron micrographs of hearts from animals treated with E2 (20 μg) plus estrogen receptor antagonist ICI 182,780 (1 mg), WAY-169916 (1 mg/kg), and ICI 182,780 or vehicle. Sarcomere structural features are altered and contraction bands are present in all groups. The markedly swollen mitochondria with disrupted cristae and osmophillic inclusion bodies indicate irreversible damage. Magnification: ×19000.

DISCUSSION

Previous studies with WAY-169916 demonstrated a reduction in infarct size when the compound was administered after the initiation of ischemia or 5 minutes before reperfusion.¹⁸ Our studies expand on the previous publication, demonstrating that pretreatment with WAY-169916 reduced tissue damage and neutrophil infiltration to the same extent as with estrogen treatment. We further confirmed the requirement of the estrogen receptor with the use of ICI 182,780 (fulvestrant, Faslodex), a non-selective ER antagonist that is devoid of known agonist effects.^19,20 Pretreatment with ICI 182,780 significantly impaired the ability of WAY-169916 to protect the rabbit heart against irreversible myocardial injury in response to regional ischemia and reperfusion. Prior experiments from our laboratory have shown that administration of the estrogen-receptor antagonist ICI 182,780 alone to the ovariectomized rabbit did not effect myocardial infarct size.⁹ The attenuation of protection by ICI 182,780 supports the concept that WAY-169916 exerts its cardioprotective activity via interaction with the estrogen receptor. To the best of our knowledge, this is the first study to demonstrate that the ability of the pathway-selective ER ligand WAY-169916 to attenuate cardiac ischemic damage is ER-dependent.

WAY-169916 functions through either estrogen receptor (ERα or ERβ) to inhibit NF-κB transcriptional activity in vitro, but it is devoid of classical estrogenic effects such as uterine stimulation, prevention of hot flushes, and prevention of bone loss in vivo.^15,21 Although both WAY-169916 and E2 can inhibit NF-κB functional activity, this is not a property of all ER ligands.^14,15 Neither the selective estrogen receptor modulator raloxifene nor the ER antagonist ICI 182,780 can promote the inhibition of NF-κB when bound to the ER.¹⁵ While raloxifene does not inhibit NF-κB activity, it has been shown to protect the myocardium in some^22,23 but not all²⁴ models of ischemia-reperfusion injury. The ability of raloxifene to protect the heart in an ER-mediated mechanism that is not dependent on NF-κB suggests that ER activation has other protective actions. It is well established that injury due to ischemia and reperfusion has an inflammatory component, and inhibitors of inflammation have been shown to reduce irreversible tissue injury that occurs after reperfusion.^25-28 WAY-169916 has been shown to be effective in several models of inflammation,^15,29 and the ability of WAY-169916 to inhibit the inflammatory response is a possible mechanism for its cytoprotective action in the present experimental model of myocardial ischemia/reperfusion injury.

The present study indicates that WAY-169916 provides a protective effect on the ischemic myocardium, reducing infarct size, and this observed protection is mediated via the estrogen receptor. Further studies must be performed to determine the effects of WAY-169916 and E2 on NF-κB activation and the inflammatory response following ischemia and reperfusion. Specifically, we need to determine the effects on cytokine and chemokines that are activated through NF-κB after ischemia and reperfusion.

This study expands on the previous studies by administering WAY-169916 before an ischemic event, which is more relevant to a woman on daily therapy. When WAY-169916 was administered before ischemia, the infarct sparing effects were far greater than previously observed when the compound was administered immediately before reperfusion.¹⁸ Future studies with chronic, oral dosing are required to more accurately mimic a clinical situation. Another necessary study is to determine the effects of WAY-169916 on infarct size in hypercholesterolemic animals, as women will likely begin therapy later in life and most likely have varying degrees of atherosclerosis and may respond differently to therapy. Most importantly, this compound has previously been shown to not possess classic estrogenic activity such as an increase in uterine growth or transcription of hepatic ER target genes.^15,21 Given that WAY-169916 lacks classical estrogenic effects, it and others in its class may be therapeutically useful in the preventive management of cardiovascular disease in post-menopausal patient.

Limitations of the Study: Relevance to Clinical Trials

Observational trials as well as extensive experimental data suggest that hormonal therapies might prevent coronary artery disease.^30-32 Despite these promising studies, recent clinical trials concluded that hormonal therapies do not confer a benefit towards the prevention of heart disease.^31,33-35 Although clinical studies did not show a benefit, our studies and those of others have suggested that estrogens are cardioprotective.^36-38 One possible explanation is that, the endpoints for the clinical trials and the reported experiments are clearly different. The clinical trials endpoints were coronary events and they examined the ability of hormone replacement to prevent adverse primary or secondary coronary outcomes.^39,40 Our studies do not attempt to show that administration of WAY-169916 will prevent an event, but rather that the presence of WAY-169916 or E2 will reduce the extent of myocardial injury secondary to ischemia-reperfusion, thereby resulting in the salvage of myocardial tissue in the jeopardized ischemic zone or area at risk. Therefore, our studies cannot conclude that the clinical trials are incorrect, but rather that when compared with an appropriate control group the extent of irreversible tissue injury that occurs upon reperfusion is decreased significantly when either estrogen (specifically E2) or the pathway-selective ER ligand WAY-169916 are present.