Catechol-O-Methyltransferase Is Dispensable for Vascular Protection ...

GENERAL

ENDOCRINOLOGY

Catechol-O-Methyltransferase Is Dispensable for Vascular Protection by Estradiol in Mouse Models of Atherosclerosis and Neointima Formation Anna S. Wilhelmson, Johan Bourghardt-Fagman, Joseph A. Gogos, Per Fogelstrand, and Åsa Tivesten Wallenberg Laboratory for Cardiovascular Research (A.S.W., J.B.-F., P.F., Å.T.), Institute of Medicine, Sahlgrenska University Hospital, University of Gothenburg, SE-413 45 Gothenburg, Sweden; and Department of Physiology and Cellular Biophysics and Department of Neuroscience (J.A.G.), Columbia University, College of Physicians and Surgeons, New York, New York 10032

Estradiol is converted to the biologically active metabolite 2-methoxyestradiol via the activity of the enzyme catechol-O-methyltransferase (COMT). Exogenous administration of both estradiol and 2-methoxyestradiol reduces experimental atherosclerosis and neointima formation, and COMT-dependent formation of 2-methoxyestradiol likely mediates the antimitogenic effect of estradiol on smooth muscle cells in vitro. This study evaluated whether 2-methoxyestradiol mediates the vasculoprotective actions of estradiol in vivo. Wild-type (WT) and COMT knockout (COMTKO) mice on an apolipoprotein E-deficient background were gonadectomized and treated with estradiol or placebo. Exogenous estradiol reduced atherosclerotic lesion formation in both females (WT, ⫺78%; COMTKO, ⫺82%) and males (WT, ⫺48%; COMTKO, ⫺53%) and was equally effective in both genotypes. We further evaluated how exogenous estradiol affected neointima formation after ligation of the carotid artery in ovariectomized female mice; estradiol reduced intimal hyperplasia to a similar extent in both WT (⫺80%) and COMTKO (⫺77%) mice. In ovarianintact female COMTKO mice, atherosclerosis was decreased (⫺25%) compared with WT controls. In conclusion, the COMT enzyme is dispensable for vascular protection by exogenous estradiol in experimental atherosclerosis and neointima formation in vivo. Instead, COMT deficiency in virgin female mice with intact endogenous production of estradiol results in relative protection against atherosclerosis. (Endocrinology 152: 4683– 4690, 2011)

urrent clinical practice widely uses estradiol (E2), the most important endogenous estrogen, as hormone replacement therapy for postmenopausal women (1). In experimental studies, E2 protects against both atherosclerosis and neointima formation (2– 6), and two randomized clinical trials investigating atherosclerosis prevention are presently evaluating E2-based hormone replacement therapy regimens (7). Thus, identifying the pathways for the vascular protective actions of E2 is vital. E2 is metabolized by the cytochrome P-450 system to hydroxyestradiols (e.g. 2-hydroxyestradiol). The enzyme catechol-O-methyltransferase (COMT) then fur-

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ther rapidly metabolizes 2-hydroxyestradiol to 2methoxyestradiol (2-ME2) (8, 9). During the last decade, this metabolite has been studied extensively because of its potent antiproliferative and antiangiogenic capacity (10 –12), and 2-ME2 is currently undergoing evaluation in clinical trials as therapy for both cancer and rheumatoid arthritis (13). Earlier studies ascribed several cardiovascular protective actions to 2-ME2, including inhibition of vascular smooth muscle cell proliferation and extracellular matrix deposition, improved endothelial function, and decreased cholesterol levels (14 –16). We have previously demonstrated that

ISSN Print 0013-7227 ISSN Online 1945-7170 Printed in U.S.A. Copyright © 2011 by The Endocrine Society doi: 10.1210/en.2011-1458 Received July 5, 2011. Accepted September 22, 2011. First Published Online October 18, 2011

Abbreviations: ApoE, Apolipoprotein E; COMT, catechol-O-methyltransferase; COMTKO, COMT knockout; E2, estradiol; I/M, intima to media; 2-ME2, 2-methoxyestradiol; P, placebo; WT, wild type.

Endocrinology, December 2011, 152(12):4683– 4690

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COMT and Vascular Protection by Estradiol

administration of 2-ME2 protects against atherosclerosis development in female mice (17), and other groups published similar results using experimental models of neointima formation and vascular remodeling (18, 19). COMT-mediated production of 2-ME2 has been demonstrated to mediate the antimitogenic effect of E2 in vitro: in a study by Zacharia et al. (20), the inhibitory effect of E2 on proliferation was abrogated in vascular smooth muscle cells from COMT knockout (COMTKO) mice. Together with observations that 2-ME2 exerts vascular protective actions similar to those of E2 (2–5, 12, 14 –19, 21, 22), that finding raises the question whether 2-ME2 mediates the protective actions of E2 on the vasculature also in vivo. To address this question, we used two different experimental models [i.e. atherosclerosis formation in apolipoprotein E (ApoE)-deficient mice and neointima formation after vascular injury] to compare the vasculoprotective effects of exogenous as well as endogenous E2 in COMTKO mice and wild-type (WT) littermates.

Materials and Methods Animals The generation of COMT-deficient mice has been described previously (23). The mutated COMT allele was introduced into a mixed 129Sv/C57BL/6J genetic background, and the mice were backcrossed more than 10 generations to a C57BL/6J background. Homozygous COMTKO mice and WT littermates were obtained by breeding heterozygous males and females. The mice were further crossed with ApoE knockout mice (C57BL/6J back-

TABLE 1.


ground, model APOE-M; Taconic Europe A/S, Lille Skensved, Denmark) to obtain COMTKO and WT mice on a homozygous ApoE-deficient background. All mice were housed in a temperature- and humidity-controlled room with a 0600- to 1800-h light cycle and consumed diet and tap water ad libitum. During surgical procedures, the mice were anesthetized (2% isoflurane in air; Baxter Medical, Kista, Sweden), anesthesia depth was monitored by limb withdrawal using toe pinching, and a postoperative analgesic was given sc (buprenorfin 50 ␮g/kg; RB pharmaceuticals, Berkshire, UK). At the end of the study, mice were euthanized during anesthesia (2% isoflurane in air; Baxter Medical); blood was drawn from the left ventricle and the heart was dissected. All procedures were approved by the Ethics Committee on Animal Care and Use in Gothenburg and conformed to the National Institutes of Health Guide for the Care and Use of Laboratory Animals (7th edition, 1996) and the directive 2010/63/EU of the European Parliament.

Genotyping Genomic DNA was isolated from tail biopsies as previously described (24). The ApoE genotype was assessed using PCR amplification of genomic DNA (protocol from Jackson Laboratory, Bar Harbor, ME). For COMT genotyping, a PCR method was developed using 5⬘-ACC ATG GAG ATT AAC CCT GAC TAC G-3⬘ (sense) and 5⬘-GTG TGT CTG GAA GGT AGC GGT C-3⬘ (antisense) primer set to detect the COMT gene (comt) allele, and 5⬘-CAT TCT GCA CGC TTC AAA AG-3⬘ (sense) and 5⬘-TGT CTG TTG TGC CCA GTC AT-3⬘ (antisense) primer set to detect the PGK-neomycin gene (neo) cassette that replaces exons 2– 4 of the COMT gene. A 500-bp (COMT) and a 170-bp (neo) fragment were generated using ReddyPCRMix (ABgene, nucleic acid amplification; Epsom, UK) in the following thermal cycles: an initial temperature of 95 C for 3 min and 35 cycles consisting of 95 C for 1 min, annealing temperature of 55 C for 30 sec, and expansion at 72 C for 1 min with a final extension at 72 C for 5

Body and organ weights, and blood pressure in WT and COMTKO mice treated with exogenous E2 WT P

Females, n Body weight, before diet (g) Body weight gain, diet period (g) Uterus (mg) Diastolic pressure (mm Hg) Systolic pressure (mm Hg) Mean arterial pressure (mm Hg) Males, n Body weight, before diet (g) Body weight gain, diet period (g) Seminal vesicles (mg) Diastolic pressure (mm Hg) Systolic pressure (mm Hg) Mean arterial pressure (mm Hg)

WT E2

P value P value P value genotype treatment interaction

COMTKO P

COMTKO E2

13 13 18.5 ⫾ 0.4 18.6 ⫾ 0.2 15.1 ⫾ 0.8 5.8 ⫾ 0.2

10 18.5 ⫾ 0.4 14.2 ⫾ 0.9

10 18.3 ⫾ 0.5 6.3 ⫾ 0.5

0.67 0.79

0.83 ⬍0.001

0.61 0.30

8.4 ⫾ 1.1 87 ⫾ 3 126 ⫾ 4 100 ⫾ 3

9.9 ⫾ 1.2 92 ⫾ 3 134 ⫾ 5 106 ⫾ 4

238 ⫾ 24.0 86 ⫾ 3 126 ⫾ 5 99 ⫾ 4

0.47 0.18 0.062 0.12

⬍0.001 0.087 0.060 0.077

0.46 0.95 0.81 0.93

10 12 18.8 ⫾ 0.4 20.3 ⫾ 0.4 12.0 ⫾ 0.7 8.4 ⫾ 0.4

13 19.1 ⫾ 0.3 14.4 ⫾ 0.9

12 21.3 ⫾ 0.3 8.5 ⫾ 0.5

0.065 0.057

⬍0.001 ⬍0.001

0.25 0.084

2.7 ⫾ 0.3 20.3 ⫾ 2.3 87 ⫾ 4 88 ⫾ 3 125 ⫾ 3 129 ⫾ 3 99 ⫾ 5 102 ⫾ 4

3.3 ⫾ 0.6 86 ⫾ 3 123 ⫾ 3 98 ⫾ 4

25.1 ⫾ 2.9 89 ⫾ 3 135 ⫾ 3 105 ⫾ 4

0.24 0.79 0.64 0.72

⬍0.001 0.37 0.09 0.19

0.35 0.65 0.39 0.51

316 ⫾ 89.7 81 ⫾ 3 116 ⫾ 4 93 ⫾ 4

Female and male WT and COMTKO mice on ApoE-deficient background were gonadectomized before puberty and fed a high-fat diet from 8 wk of age until the study ended at 16 wk of age. Values represent mean ⫾ SEM. Data were analyzed by two-way ANOVA.


min. Amplified DNA fragments were visualized using ethidium bromide staining under UV light after electrophoresis in 1% agarose gel.

Study protocol for experimental atherosclerosis Three-week-old male and female WT and COMTKO mice on ApoE-deficient background were bilaterally gonadectomized and sc implanted with a slow-release pellet containing E2 (0.83 ␮g/d) or placebo (P) (60 d release; Innovative Research of America, Sarasota, FL). A new hormone-releasing pellet (E2 or P; 60 d release) was implanted sc 52 d later to ensure hormone therapy throughout the study. After weaning, the mice consumed a soyfree diet (R70; Lantma¨nnen, Stockholm, Sweden) for 5 wk, followed by a high-fat diet (no. 821424, 21% fat from lard: 0.15%

A

P

E2

WT

B

E2

Pgenotype = 0.44 Ptreatment < 0.001 Pinteraction = 0.88

4 3

WT Placebo WT E2 COMTKO Placebo COMTKO E2

2 1 0 WT

C % Lesion area

4

cholesterol; Special Diets Services, Essex, UK) from 8 wk of age to accelerate the formation of atherosclerotic plaques. After 8 wk on high-fat diet, 16-wk-old mice were anesthetized with isoflurane (Baxter Medical). A Samba transducer catheter (Samba Sensors, Gothenburg, Sweden) was placed in the left carotid artery to measure diastolic, systolic, and mean arterial blood pressure. Data were collected using a PowerLab data acquisition unit with Powerlab 5.0 (ADInstruments, Sydney, Australia), and the data were averaged over a 2-min period after stabilization of the arterial pressure. The mice were fasted 3 h before blood was drawn from the left ventricle, and the circulatory system was perfused with 0.9% saline (pH 7.4) under physiological pressure. The aorta was dissected from the heart to the iliac bifurcation and fixed in 4% paraformaldehyde for subsequent en face analysis. Organs were dissected and the wet weight was recorded. In a separate study, gonadal-intact male and female ApoEdeficient WT and COMTKO mice consumed a soy-free diet (R70; Lantma¨nnen) until 8 wk of age and then consumed a highfat diet for 8 wk (no. 821424, 21% fat from lard: 0.15% cholesterol; Special Diets Services) to accelerate the formation of atherosclerotic plaques. At 16 wk of age, serum and tissue were collected.

The vessel was dissected free from adipose and connective tissue, cut open longitudinally, and pinned flat on silicone-coated dishes. The aortas were stained with Sudan IV for lipids, and digital images of the entire vessel were captured. The outline of the aortic surface was defined manually, whereas the stained lesion area was defined using computerized color selection in BioPix iQ 2.2.1 (Gothenburg, Sweden). The extent of atherosclerosis was calculated as the percentage of the aortic surface covered by lesions.

Serum lipid quantification

COMTKO

♂

5

4685

En face analysis of the aorta

COMTKO

♀

5

% Lesion area

P

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Pgenotype = 0.55 Ptreatment < 0.01 Pinteraction = 0.55

Total cholesterol and triglycerides in serum were quantified using Infinity reagents (cholesterol no. TR13421 and triglycerides no. TR22421; Thermo Fisher Scientific, Pittsburgh, PA).

Study protocol for carotid artery injury model

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WT Placebo WT E2 COMTKO Placebo COMTKO E2

2 1 0 WT

COMTKO

FIG. 1. Atherosclerotic lesion area in en face preparation of the aorta. Female and male WT and COMTKO mice on ApoE-deficient background were gonadectomized before puberty and treated with exogenous E2 (0.83 ␮g/d) or placebo. Between 8 and 16 wk of age, the mice consumed a high-fat diet. At 16 wk of age, lesion formation was assessed in the aortas mounted en face. A, Representative photographs showing typically sized atherosclerotic lesions from female mice. B and C, Lesion area showing positive lipid staining with Sudan IV, expressed as the percentage of the total vessel area in females (WT P, n ⫽ 13; WT E2, n ⫽ 13; COMTKO P, n ⫽ 10; COMTKO E2, n ⫽ 10) (B) and males (WT P, n ⫽ 10; WT E2, n ⫽ 12; COMTKO P, n ⫽ 13; COMTKO E2, n ⫽ 12) (C). Values represent mean ⫾ S⌭⌴. Data were analyzed by two-way ANOVA.

The mice consumed a soy-free diet (R70; Lantma¨nnen) throughout the study. At 9 wk of age, female WT and COMTKO mice were bilaterally ovariectomized and sc implanted with a slow-release pellet containing E2 (0.83 ␮g/d) or P (60 d release; Innovative Research of America). At 10 wk of age, carotid injury was introduced as described previously (25). Briefly, the mice were anesthetized with isoflurane (Baxter Medical), and the right common carotid artery was exposed through a midline cervical incision and ligated with a 6-0 silk suture just proximal to the carotid bifurcation. Twenty-eight days after injury, the circulatory system was perfused with 0.9% saline (pH 7.4) followed by 4% paraformaldehyde under physiological pressure and the carotid artery was excised. In a separate study, 10-wk-old gonadal-intact male and female WT and COMTKO mice sustained a carotid injury, as described above. Throughout the study, the mice consumed a soy-free diet (R70; Lantma¨nnen). The mice were euthanized 28 d after injury and the carotid artery was excised.

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COMT and Vascular Protection by Estradiol

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A Intimal area (mm2)

0.05

Pgenotype = 0.65 Ptreatment