Abnormal Vascular Function and Hypertension in Mice Deficient in Estrogen Receptor Yan Zhu, Zhao Bian, Ping Lu, Richard H. Karas, Lin Bao, Daniel Cox, Jeffrey Hodgin, Philip W. Shaul, Peter Thorén, Oliver Smithies, Jan-Åke Gustafsson, and Michael E. Mendelsohn

Ring Studies: All animal care and procedures were approved by the Tufts University-New England Medical Center Animal Research Committee. All ER-deficient mice and WT littermates described in this report are from the mice created in the Smithies laboratory (1), recently designated ERKO_CH (2) and then bred in North Carolina (Fig. 4, manuscript) or Sweden (Web fig. 2), as indicated. All animals genotyped by PCR of tail DNA. Mouse thoracic aortas were removed, cleaned of fat and connective tissues, cut into 3 mm rings, and incubated in organ baths containing 2 ml Krebs buffer (mmol/L: NaCl 120, KCl 4.7, CaCl₂ 2.5, MgSO₄ 1.2, KH₂PO₄ 1.2, NaHCO₃ 25 and glucose 5.5, pH 7.4) at 37°C with a 5%CO₂-95%O₂ atmosphere until use. Vessels were equilibrated for 60 min with changes of buffer every 20 minutes. For pretreatment with E2 and/or other compounds, rings were incubated at 37°C for 18-20 hours with the compound(s) under investigation. Endothelial-denuded (-En) or endothelial-intact (+En) rings were studied in Figures 1 through 3, as noted. Denudation of endothelium was performed by gently passing a 0.65 mm guide wire through the vessel lumen. Any adventitia observed was also removed. Loss of endothelium in all -En rings studied was confirmed by loss of relaxation to Ach (10^-6 M). Isometric tension studies were performed using force transducers connected to PowerLab/800 Eight-channel 100 kHz A/D converter (AD Instruments, Mountainview, CA). For studies of +En rings, optimal resting tension was determined in baseline studies, vessels were submaximally precontracted with PE (to 80% maximal; typically 1 × 10^-6 M), and endothelial function was evaluated by vascular relaxation to Ach. For -En studies, cumulative dose-response curves to PE (10^-9 to 3 × 10^-6 M) were established. At the end of each experiment, all + En vessels were again contracted by PE and for both +En and -En rings, relaxation to SNP (10^-10 to 10^-7 M) was tested to verify that baseline contractile function and endothelium-independent relaxation were preserved during the course of the study. Transgenic mice deficient in iNOS (iNOS^-/-) and WT controls were purchased from the Jackson Laboratory (Bar Harbor, ME) (3). Data were collected and analyzed using Chart for Windows (AD Instruments, Mountainview, CA). Values are means ± SEM and n indicates the number of observations. Comparisons were tested with ANOVA, followed by Student-Newman-Keuls test. A difference was considered significant at P < 0.05.

Transient Transfection Studies. Cells were harvested for luciferase assays 48 hours following transfection. Total DNA transfected was held constant by including backbone plasmid DNA as necessary. All results presented are normalized for the activity of a co-transfected lacZ expression plasmid by standard -galactosidase assay.

Immunoblotting Endothelial-denuded (or endothelial-intact for inset, Fig. 2B), aortas were homogenized in extraction buffer [in mmol/L: NaCl 154, tris(hydroxymethyl)-aminomethane (Tris) base 20, EDTA 10, sodium vanadate 10, and sodium dodecyl sulfate (SDS) 2%, pH 7.4]. Homogenates were centrifuged for 20 min at 10,000 g at 4°C and the supernatant was removed and used for Western blot analysis. Protein concentrations were determined using the D_c Protein assay kit (Bio-Rad, Hercules, CA) and/or Ponceau staining of nitrocellulose membranes to assure equal loading (20 micrograms per lane). Membranes were then incubated with monoclonal or polyclonal antibodies specific for eNOS, iNOS, or nNOS (Transduction Laboratories, Lexington KY) and developed with horseradish peroxidase secondary antibodies and ECL.

Electrophysiologic Methods Currents were recorded from mouse aorta smooth muscle cells grown in culture from explants, as previously described (4). Both WT and ERKO cells were cultured at the same time, using the same techniques, and they were passaged the same number of times before plating for electrophysiological recordings. Recordings were done in the whole-cell configuration of the patch-clamp technique with an Axon 200B patch-clamp amplifier and a Macintosh-base computer system running HEKA "Pulse" acquisition software. All recordings were made at room temperature (23°-25°C) with borosilicate glass (VWR). Electrode tips were between 1 and 2 Mohms; they were dipped in Sticky Wax (Emeryville, CA) and fire-polished before use. Data were filtered at 5 KHz and leak subtracted with a P/5 protocol whose holding potential was -50 mV. The membrane potential was also held at -50 mV before and after voltage-protocols (as indicated) were applied. Solutions were as follows: Pipette: 140 mM KCL, 10 mM Hepes, 5 mM EGTA, 2 mM MgATP, and 0.3 mM Na₂GTP. Bath: 125 mM NaCl, 5 mM KCl, 20 mM Hepes, 5 mM EGTA, 2 mM MgCl₂, 5 mM CaCl₂ and 10 mM Glucose. Mean current densities for WT and ERKO VSMC, calculated as the average of each cell's current amplitude divided by its membrane capacitance, the later of which was determined with the patch-clamp amplifier's whole-cell capacity-compensation circuitry.

Direct Hemodynamic Monitoring Cannulation method Nineteen male mice weighing 26 to 36 g and 20 female mice weighing 22 to 28 g were included in this study. The study shown was approved by the regional Animal Ethical Committee of the Karolinska Institute. Mice were anaesthetized with 2% isofluran (Abbott Lab Ltd, Kent, England). The right carotid artery was cannulated with a PE-10 catheter, which was tunneled subcutaneously to exit at the back, and connected to a swivel tether system (custom made). Sterile saline was infused continuously via the cannula (2 to 3 l/min). All mice recovered completely from anaesthesia within a few minutes after the termination of isofluran. The mean arterial BP (MAP) in conscious, freely-moving mice was started to be recorded (Grass Polygraph model 7) at 1 hour post-operation, and it lasted for 24 h (twice per min), consisting of a cycle of 6 h light, 12 h dark,and 6 h light.

Telemetric method Implantable, miniaturised mouse blood pressure transmitters (Data Sciences International, St Paul, MN) were used to directly measure arterial pressure digitally from within individual animals (5). These devices are thought to provide the most sensitive and accurate method available at present to monitor blood pressure in conscious, freely-moving mice (5). Radiofrequency signals from the transmitters are converted to serial bit streams and data are collected and stored to disk using the Dataquest A.R.T. data acquisition system (Data Sciences International, St Paul, MN). Ambient barometric pressure is also measured and subtracted from the telemetered pressure by the Dataquest A.R.T. software to compensate for changes in atmospheric pressure. Sodium intake per 24 hours equivalent to approximately 1.3 g per day for humans, based on caloric intake (Diet = Harlan Teklad, Madison, WI).

Supplemental Results

Contraction and relaxation in response to phenylephrine were identical in intact vascular rings from ERKO mice or their WT littermates and were not altered by E2 treatment (Web fig. 1, A and B). Treatment with E2 caused an increase in eNOS protein expression in endothelium-intact WT rings, as reported previously in other animals (6, 7), and this increase was inhibited by the ER antagonist ICI 182,780 (inset, Fig. 1A). However, contraction of endothelium-intact mouse aortic rings is not altered by exposure to E2 (8). These data support that endothelial function is intact in WT and ER-deficient mice, and do not reveal any differences in the vasoactive response between the two genotypes in the absence or presence of estrogen.

Supplemental electrophysiologic data In response to voltage ramps, 3 of 8 WT cells displayed inward currents at negative potentials that rectified near the potassium equilibrium potential (E_K = -85 mV), strongly suggesting the presence of inward rectifier potassium channels (Fig. 4A, lower panel). The loss of outward current we have observed could in principal be due to the loss of either voltage-activated (K_v type), or Ca⁺² and voltage-activated (BK_Ca type), potassium channels, both of which have been implicated in the control of vasomotor tone (9). In particular, one might expect that the loss of either of these channel types would lead to a loss of feedback control over processes such as the development of myogenic tone, which involve membrane depolarization and subsequent vasoconstriction (10, 11). Our preliminary experiments with tetraethylammonium applied externally, however, suggest that the majority of the outward current in both WT and ERKO aortic smooth muscles cells is not BK_Ca, Thus it is likely the regulation of K_v channel expression that we are observing. In addition, inwardly-rectifying potassium channels are thought to in part control the smooth-muscle resting potential at low transmural pressures (9). Thus, the loss of these channels could also contribute to a hypercontractile smooth muscle phenotype, and together the loss of both channel types might contribute to the hypertension observed in ERK0 mice. It is as yet unclear, however, how alterations in ion channel expression relate to the observation that estrogen potentiates contractility in endothelial-denuded ERKO aortic rings. One might suppose that there are opposing actions of ER and ER, such that the loss of ER allows a constricting influence of ER to emerge. Pursuing this idea further, one action of ER could be to promote expression of genes important to potassium channel activation, as has been observed for BK_Ca channels via a NO/cGMP pathway (12, 13). Whether such a pathway exists for K_V channels remains to be determined.

Supplemental BP data BP became elevated in approximately half of a cohort of 20 ERKO mice followed in Sweden. At 6 to 7 months of age, direct hemodynamic measurement showed elevation of mean arterial pressure (MAP) was present in the majority of ERKO mice of each gender (Web fig. 2). Within the first 1-3 hours of a light cycle, MAP was slightly above baseline as mice were adapting to the experimental conditions. After entering the dark cycle, most of the mice became gradually more active, with an additional increase of MAP recorded during this period (12 h dark cycle). All mice became less active during the following 6-h light cycle, though MAP declined only partially back towards baseline. MAP in male ERKOs (137 ± 4.6 mm Hg, n=9) was significantly higher (P < 0.01) than that in WT males (109 ± 2.7 mm Hg, n=10), or female ERKOs (123 ± 4.9 mm Hg, n = 10). MAP in female ERKOs was significantly increased (P <0.01) compared to that of WT females (106 ± 2.9 mm Hg, n=10). MAP in heterozygous mice at 6 to 7 months of age tended to be higher than that in WT littermates, but these differences did not reach statistical significance (14). Histological examination of organs, obtained from both male and female 6- to 7-month-old ERKOs, including heart, lungs and kidneys, did not reveal any obvious pathological alterations, such as hypertrophy or fibrosis (14).

Supplemental Figure 1. Endothelial-dependent relaxation and constriction in untreated intact vascular rings from ERKO mice and WT littermates. (A) Phenylephrine concentration-constriction curves for endothelium-intact (+En) aortic rings from WT (ER^+/+, closed circles) or ERKO (ER^-/-, open circles) mice. (B) Vascular relaxation in response to acetylcholine (Ach) for endothelium-intact aortic rings from WT (closed circles) or ERKO (open circles) mice. N = 8, p = NS for WT vs. ERKO for bothPE and Acetylcholine response. Inset, expression of endothelial NOS in endothelial-intact rings from WT mice in the absence or presence of E2 (10^-8 M) or E2 + ICI-182,780 (10^-7 M) for 24 hours. One of 4 similar experiments shown.

Figure 1A

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Figure 1B

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Figure 1C

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Supplemental Figure 2. Blood pressure in conscious, freely-moving WT and ERKO mice at 6 months of age. Mean arterial pressure (MAP) from 24 h of recording in male and female 6-month-old animals (30 sample times per hour). Closed circles, WT females; closed triangles, WT males; open circles, ERKO females; open triangles, ERKO males. N = 9 to 10 in each group shown. Sodium intake per 24 hours equivalent to approximately 2.5 g per day for humans, based on caloric intake; diet = Beekay, Bantan & Kingman, Ltd., Hull, UK. All data are presented as mean ± SEM. Experiments performed using cannulation method at Karolinska Institute, Sweden by Dr. Zhao Bian.

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Supplemental Figure 3. Heart rates in conscious, freely-moving 12-month-old WT and ERKO mice. Telemetric measurement of heart rate in the male ERKO mice (open circles) and littermate controls (closed circles), recorded simultaneoulsy with the blood pressure data shown in manuscript Fig. 4. Mean ± SEM for N = 5 mice per group.

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