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Gender difference of atorvastatin's vasoprotective effect in balloon-injured rat carotid arteries
European Journal of Pharmacology 553 (2006) 263 – 268 www.elsevier.com/locate/ejphar
Gender difference of atorvastatin's vasoprotective effect in balloon-injured rat carotid arteries Daisuke Kurumazuka a , Tatsuhiko Mori b,⁎, Noriko Matsumoto a , Hisashi Shirakawa a , Sayaka Kimura a , Daisuke Nakano a , Tetsuya Hayashi b , Yasushi Kitaura b , Yasuo Matsumura a b
a Department of Pharmacology, Osaka University of Pharmaceutical Sciences, Japan Department of Medicine III, Osaka Medical College, 2–7 Daigakumachi, Takatsuki, Osaka, 569-8686, Japan
Received 27 June 2006; received in revised form 14 September 2006; accepted 19 September 2006 Available online 27 September 2006
Abstract The effect of estrogen on neointimal formation in injured rat arteries has been reported to be a sexual dimorphic effect. Recently, it has been reported that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exhibit vasoprotective effects, which are independent of their cholesterol-lowering effects. In this study, we examined the gender differences of atorvastatin's effect on neointimal formation in balloon-injured rat arteries. Male and female Sprague Dawley rats underwent gonadectomy and balloon injury of the carotid artery. Ovariectomized female, as well as intact and castrated male, rats exhibited marked neointimal formation. Treatment with atorvastatin significantly reduced neointimal formation at day 14 (14 days after injury) and NADPH oxidase-dependent superoxide production at day 2 in ovariectomy, but not in intact and castrated males. In ovariectomized rats, 7 days of atorvastatin treatment from days −3 to 3 but not from days 7 to 14 suppressed neointimal formation at day 14. In this study, we showed that atorvastatin's effect on neointimal formation was female-specific and was more marked in ovariectomized female rats. NADPH oxidase-dependent superoxide production may be involved in the mechanism of the sexual dimorphic response seen in response to atorvastatin treatment. Furthermore, the results suggest the importance of treatment in the early phase after vascular injury. © 2006 Elsevier B.V. All rights reserved. Keywords: Gender difference; Statin; NADPH-dependent superoxide production; Vasoprotection
1. Introduction In the past, the cardiovascular protection thought to be offered by hormone replacement therapy (Barrett-Connor, 1997; Grady et al., 1992; Stampfer et al., 1991) for postmenopausal women was primarily attributed to the improvement of the serum lipid profile. However, recent reports have suggested that estrogen has direct protective effects, including increasing nitric oxide production, suppressing adhesion molecule activity, suppressing smooth muscle proliferation and migration, and suppressing superoxide production (Florian et al., 2004; Mendelsohn and Karas, 1999; Miller et al., 2003; Ranki et al., 2002; Tolbert and Oparil, 2001). Nevertheless, the Heart Estrogen–Progestin Replacement Study (HERS) (Hulley et al., ⁎ Corresponding author. Tel.: +81 72 683 1221; fax: +81 72 684 6598. E-mail address: [email protected] (T. Mori). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.09.036
1998) and the Women's Health Initiative Clinical Trial and observational study (WHI) (Rossouw et al., 2002) did not show any benefit of hormone replacement therapy. Furthermore, WHI indicated that the hormone replacement therapy was associated with significant increases in the incidences of breast cancer and thromboembolic diseases (Rossouw et al., 2002). Like estrogen, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert a vasoprotective effect by improving the serum lipid profile and by anti-inflammatory mechanisms that are independent of their lipid-lowering effect (Schonbeck and Libby, 2004). Increased arterial superoxide production generated by vascular NADPH oxidase has been reported to play a critical role in the pathogenesis of atherosclerosis (Zalba et al., 2005). Both estrogen and statins have been reported to exhibit anti-oxidative effects through suppression of NADPH oxidase (Florian et al., 2004; Wassmann et al., 2001). Recent reports have shown that estrogen has a
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Fig. 3. Intima to media ratios (mean ± S.E.M.) of carotid arteries at 14 days after balloon injury. The intima to media ratios were increased in ovariectomized female compared to intact females, and the ratios were significantly suppressed by atorvastatin. However, in intact and castrated males, the increase in neointimal formation was not suppressed by atorvastatin. Open bar; vehicle treatment, and hatched bar; atorvastatin treatment. ⁎P b 0.05, compared with vehicle in each. #P b 0.05 compared with intact female rats.
Fig. 1. Light micrographs of neointimal formation in balloon-injured carotid arteries of female rats at 14 days after balloon injury. The neointimal formation observed in ovariectomized female + vehicle was more prominent than in intact females. Atorvastatin treatment significantly suppressed neointimal formation.
vasoprotective effects of atorvastatin; 3) short-term treatment with atorvastatin has a long-term treatment effect; and 4) differences in NADPH oxidase activity underlie the gender difference in atorvastatin's effects. 2. Methods
sexual dimorphic vasoprotective pattern and that female rats have a more favorable treatment effect than male rats in the balloon-injured rat carotid artery model (Tolbert and Oparil, 2001). The mechanisms for the gender difference have not been fully elucidated, though a study did show that the estrogeninduced inhibition of the vascular injury response was mediated by molecular events that occur early after injury (Mori et al., 2000). In the present study, we examined whether: 1) atorvastatin treatment suppresses neointimal formation after balloon injury in rat carotid arteries; 2) there is a gender difference in the
2.1. Animal preparation Ten-week old male and female Sprague Dawley rats were obtained from Japan SLC, Inc., (Shizuoka). The experimental protocols and animal care methods were approved by the Experimental Animal Research Committee at Osaka University of Pharmaceutical Sciences. Under anesthesia using an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg), the rats either had gonadectomy (ovariectomy for female rats and castration for male rats) or sham surgery. A week later, daily oral administration of atorvastatin (3 mg/kg/day in 0.5% sodium Table 1 Effects on myointimal proliferation after balloon injury of carotid arteries of female intact and ovariectomized rats of atorvastatin 3 mg·kg− 1·d− 1 and vehicle 2 mL·kg− 1·d− 1 treatment for 14 days
Fig. 2. Light micrographs of neointimal formation in balloon-injured carotid arteries of male rats at 14 days after balloon injury. Prominent neointimal formation is observed in the intact castrated male. Atorvastatin treatment could not suppress neointimal formation in intact castrated males.
Intact females
Ovariectomized females
Vehicle
(n = 6)
(n = 6)
Body weight, g Area of media, mm2 Area of intima, mm2 Intima to media ratios
218 ± 4 0.131 ± 0.003 0.133 ± 0.007 1.019 ± 0.038b
244 ± 6a 0.127 ± 0.008 0.166 ± 0.018 1.288 ± 0.081a
Atorvastatin
(n = 4)
(n = 8)
Body weight, g Area of media, mm2 Area of intima, mm2 Intima to media ratios
231 ± 2 0.147 ± 0.004 0.146 ± 0.009 0.992 ± 0.052b
253 ± 3a 0.122 ± 0.003 0.123 ± 0.008b 1.000 ± 0.050b
Morphometric analysis of rat carotid arteries 14 days after balloon injury in intact and ovariectomized female rats. Each value represents the mean ± S.E.M. aP b 0.05 compared with intact females with vehicle. b P b 0.05 compared with ovariectomized females with vehicle.
D. Kurumazuka et al. / European Journal of Pharmacology 553 (2006) 263–268 Table 2 Effects on myointimal proliferation after balloon injury of carotid arteries of male intact and castrated rats of atorvastatin 3 mg·kg− 1·d− 1 and vehicle 2 mL·kg− 1·d− 1 treatment for 14 days Intact males
Castrated males
Vehicle
(n = 8)
(n = 8)
Body weight, g Area of media, mm2 Area of intima, mm2 Intima to media ratios
369 ± 4 0.142 ± 0.005 0.159 ± 0.012 1.110 ± 0.040
349 ± 6 0.154 ± 0.006 0.176 ± 0.019 1.126 ± 0.082
Atorvastatin
(n = 5)
(n = 7)
Body weight, g Area of media, mm2 Area of intima, mm2 Intima to media ratios
374 ± 8 0.134 ± 0.007 0.157 ± 0.022 1.116 ± 0.105
348 ± 7 0.149 ± 0.006 0.179 ± 0.011 1.195 ± 0.040
Morphometric analysis of rat carotid arteries 14 days after balloon injury in intact and castrated male rats. Each value represents the mean ± S.E.M.
carboxymethylcellulose (CMC-Na)), supplied by Pfizer, or vehicle (0.5% CMC-Na in water) was started. In most of the rats, the administration of atorvastatin or vehicle was started 3 days before (day − 3) and continued until 14 days after balloon injury (day 14). Furthermore, some of the ovariectomized female rats were divided into 4 groups and treated with atorvastatin or vehicle as follows: 1) atorvastatin from day − 3 through day 14; 2) atorvastatin from day − 3 to day 3; 3) atorvastatin from day 7 to day 14; and 4) vehicle treatment from day − 3 to day 14. Neointimal formation was evaluated at day 14.
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using ovariectomized female rats sacrificed at days 1, 2, 3, 7, and 14. Given the results of this protocol, we then decided to measure the NADPH-dependent superoxide production in all groups 2 days after balloon injury. Both injured (right) and uninjured (left) common carotid arteries were cleared of adherent adipose and loose connective tissue in situ and were harvested in ice-cold modified Krebs–HEPES buffer containing (in mmol/L): NaCl 99.01, HEPES 20, KCl 4.69, MgSO4 0.59, KH2HPO4 1.03, NaHPO4 25, CaCl2 1.41, and glucose 11.1 (pH 7.4). The tissues were then gently flushed with cold buffer to remove blood from the lumen and were cut into 3 segments. The segments were incubated with NADPH (100 μM) in buffer at 37 °C for 15 min s. Lucigeninenhanced chemiluminescence was measured in 2 mL Krebs– HEPES buffer containing lucigenin (5 μmol/L) using a Berthold FB12 single-tube luminometer, modified to maintain a sample temperature of 37 °C. Chemiluminescence was measured continuously for 15 min s after allowing for dark adaptation and was expressed as relative light units per minute per milligram vessel dry weight (RLU/min/mg). 2.5. Statistical analysis All values are expressed as mean ± S.E.M. The data were statistically analyzed using one-way analysis of variance combined with Dunnett's multiple range test for multiple comparisons. Differences were considered significant when the P value was less than 0.05. 3. Results 3.1. Response of right carotid arteries to balloon injury
2.2. Balloon injury procedure Rats were anesthetized using intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg), and the right carotid artery was injured with a 2F Fogarty balloon catheter (Baxter International, Deerfield, IL, USA) (Mori et al., 2000). The left carotid artery was not damaged. 2.3. Morphometric analysis
Ovariectomized female rats showed an increased body weight compared to intact female rats. Castration had no effect on the body weight of male rats. Atorvastatin treatment itself had no effect on the body weight of male and female rats. Neointimal formation was more marked in ovariectomized female rats than in intact female rats. In male rats, significant neointimal formation was also observed, which was not affected by castration. Atorvastatin significantly reduced neointimal formation
Two weeks after balloon injury of the right carotid artery, the rats were sacrificed with a sodium pentobarbital overdose (75 mg/kg) and perfused with 10% formalin to fix the arteries. Morphometric analysis of each arterial segment was performed with a computer-based Motic Image Plus 2.0 Morphometric system (Shimadzu, Kyoto). The degree of neointimal formation of the injured carotid artery was expressed as the absolute area of neointima and the intima to media ratios. 2.4. NADPH oxidase activity NADPH oxidase activity was measured based on the degree of NADPH-dependent superoxide production in the isolated carotid arteries assessed by a lucigenin-enhanced assay as previously described (Pu et al., 2003). At first, we examined the time course of NADPH-dependent superoxide production after balloon injury
Fig. 4. The time course of NADPH-dependent superoxide production after balloon injury using ovariectomized female rats sacrificed at days 1, 2, 3, 7, and 14. NADPH-dependent superoxide production in injured carotid arteries began to increase at day 2; this increase continued through day 7, and then returned to the level of uninjured carotid arteries at day 14. Each value represents the mean ± S.E.M. ⁎P b 0.01, compared with sham.
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Fig. 5. Superoxide production depended on the level of NADPH oxidase in uninjured and injured carotid arteries 2 days after balloon injury. The production in injured carotid arteries was increased more than in uninjured carotid arteries. As well, the increase in injured arteries was more prominent in ovariectomized female rats, male intact rats, and castrated male rats, compared to intact female rats. Atorvastatin treatment suppressed the production in ovariectomized female rats but not in male (intact or castrated) rats. Each value represents the mean ± S.E.M. Open bar; vehicle treatment, and hatched bar; atorvastatin treatment. ⁎P b 0.01, compared with ovariectomized female + vehicle.
in ovariectomized female rats (Fig. 1) but not in male (intact or castrated) rats (Fig. 2). In intact females, neointimal formation was not affected by atorvastatin. Morphometric analysis showed that the neointimal area and the intima to media ratios were attenuated by atorvastatin in ovariectomized female rats, but not in male (intact or castrated) rats. (Fig. 3, Tables 1 and 2). 3.2. NADPH-dependent superoxide production in injured and uninjured carotid arteries NADPH-dependent superoxide production in injured carotid arteries began to increase at day 2; this increase continued through day 7, and then returned to the level of uninjured carotid arteries at day 14 (Fig. 4). NADPH-dependent superoxide production in the uninjured carotid arteries was similar in all groups, and atorvastatin did not affect basal NADPH-dependent superoxide production in uninjured arteries (Fig. 5). In injured carotid arteries, NADPH-dependent superoxide production was increased in all rats, but the increase was less in intact female rats than in the other groups. Treatment with atorvastatin significantly suppressed NADPH-dependent superoxide production in
Fig. 6. Intima to media ratios (mean ± S.E.M.) of balloon-injured carotid arteries of ovariectomized female rats treated with atorvastatin or with vehicle for the time periods indicated. Short-term treatment from day − 3 to day 3 but not from day 7 to day 14 shows similar neointimal suppression to treatment from day −3 to day 14. ⁎P b 0.05 compared with vehicle.
the injured arteries of ovariectomized female rats but not in the arteries of male (intact or castrated) rats (Fig. 5). 3.3. Time-window response of balloon-injured right carotid arteries in rats Neointimal formation was reduced by 26% with atorvastatin treatment from day − 3 to day 14 (Fig. 6). Seven-day treatment with atorvastatin from day − 3 to day 3 also suppressed neointimal formation at day 14, with a 23% inhibition that was almost the same as that seen with day − 3 to day 14 treatment. However, 7-day treatment starting at day 7 did not suppress neointimal formation. 4. Discussion The major findings of the present study are: 1) atorvastatin treatment markedly attenuated neointimal formation in ovariectomized female rats but not in male (intact or castrated) rats; 2) treatment with atorvastatin significantly decreased NADPHdependent superoxide production in ovariectomized female rats but not in male (intact or castrated) rats; and 3) short-term atorvastatin treatment from day −3 to day 3 reduced neointimal formation to a level comparable to that seen with continuous atorvastatin treatment given from 3 days before to 14 days after injury. These results suggest that atorvastatin attenuates neointimal formation in balloon-injured vessels by reducing the production of oxidative stress. Most importantly, atorvastatin's effect was observed only in female rats, suggesting that there may be a marked gender difference in the mechanism of atorvastatin's vasoprotective effect. Gender differences in the vasoprotective effects of statins have not been shown in clinical studies. In these studies, patients with a high cholesterol level were recruited, and the vasoprotective effect of statin treatment was considered to have been the result of the lipid lowering effect rather than any other effects. In our experiment, the dosage of atorvastatin (3 mg/kg/
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day) used had no effect on the lipid profile (Chen et al., 2003). Therefore, the gender difference in atorvastatin's vasoprotective effect in this study was not dependent on the lipid-lowering effect of atorvastatin. Thus, our results appear to differ from the results of clinical studies. However, recently, Seed showed that in the three randomized controlled trials, the Scandinavian Simvastatin Survival Study (4S), the Cholesterol And Recurrent Events study (CARE) and The Long-term Intervention with Pravastatin in Ischaemic Disease (LIPID), women accounted for approximately 20% of the subjects in each study, cardiovascular events were fewer in women on active medication but in no single study were there sufficient numbers to result in a significant reduction in cardiovascular or total mortality, even in the 8-year follow up of the 4S (Seed, 2002). This recent report suggests that there is a gender difference in the effect of statins on cardiovascular events in clinical trials. Interestingly, the gender-dependent effect of atorvastatin is very similar to the vasoprotective effect attributed to estrogen. Previous results (Tolbert and Oparil, 2001) and our present results demonstrate that: 1) gonadectomy of female rats was associated with a more robust neointimal proliferative response to balloon injury; and 2) gonadectomy of male rats did not alter the neointimal response. These results suggest that estrogen is an important factor for decreased neointimal formation in intact female rats. Indeed, it has been shown that estrogen administration to ovariectomized female rats attenuated neointimal formation (Tolbert and Oparil, 2001). Jovanovic showed the pregnancy associated hypotrophy of carotid and thoracic endothelial and smooth muscle cells in guinea pig (Jovanovic and Jovanovic, 1997, 1998). These results suggest that the estrogen effects on arterial remodeling not only in injured but also intact artery. To our knowledge, there are no reports suggesting that there is a gender difference in the metabolism of atorvastatin in rats or that atorvastatin's metabolism is affected by sex hormones to a large extent. Therefore, the apparent lack of effect of atorvastatin in male rats could not be due to a reduced plasma concentration of atorvastatin in male rats. Some studies of postmenopausal women found that statin treatment in addition to hormone replacement therapy had additive cholesterol lowering (Dobrzycka et al., 2003; Koh et al., 1999) and antioxidative effects (Seeger et al., 2000), as well as providing additional cardiovascular risk reduction (Herrington et al., 2002). These effects may be partially the result of the cholesterol lowering effects, but Mueck et al. reported that the estrogen/statin combination had a beneficial effect on biochemical markers of endothelial function in human coronary artery cell cultures (Mueck et al., 2001). Furthermore, statins have been reported to inhibit the proliferation of estrogen receptor-negative human breast cancer cells (Mueck et al., 2003). Therefore, the direct vasoprotective mechanisms of estrogen and statins appear to be somewhat different; the effect of statins is not mediated by the estrogen receptor. In this study, we could not examine the effect of an estrogen receptor blocker combined with statin treatment. Nevertheless, the statin's vasoprotective effect seems to be positioned downstream to the estrogen receptor in this receptor signaling pathway. Oxidative stress plays a crucial role in the pathogenesis of atherosclerotic vascular disease (Harrison
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et al., 2003). Recently, reactive oxygen species have also been implicated in other pathological processes that occur in the vessel wall, including endothelial dysfunction, activation of matrix metalloproteinases, and vascular smooth muscle cell (VSMC) migration, growth, and apoptosis (Taniyama and Griendling, 2003). Increased NADPH oxidase and reactive oxygen species have been reported in coronary arteries after balloon injury (Shi et al., 2001). Overall, oxidative stress appears to play many important roles in the pathogenesis of vascular diseases, including the neointimal formation of injured arteries (Zalba et al., 2005). We also found that: 1) superoxide production generated by vascular NADPH oxidase was increased in injured carotid arteries compared with uninjured carotid arteries; 2) superoxide production in injured arteries was further increased by gonadectomy in female but not in male rats; and 3) treatment with atorvastatin significantly decreased superoxide production in ovariectomized female rats but not in male (intact or castrated) rats. Since superoxide production levels appear to be correlated with the extent of neointimal formation, NADPH oxidase and its product, superoxide, may be involved to a large extent in neointimal formation in injured arteries. Our results also suggest that atorvastatin attenuates superoxide production by NADPH oxidase in injured arteries. Independent of their lipid-lowering effects, statins have been shown to reduce oxidative stress and gene expression of p22phox, an essential subunit of NADPH oxidase, which is present in the vasculature (Itoh et al., 2002; Wassmann et al., 2001). The molecular mechanism underlying atorvastatin's effect is still unclear. Given our present data, estrogen appears to maintain a low level of NADPH-dependent superoxide production in balloon-injured carotid arteries, and when given to ovariectomized rats, atorvastatin reduced the exacerbation of NADPH-dependent superoxide production through its antioxidative properties. In this study, we could not determine why atorvastatin was ineffective in suppressing NADPH-dependent superoxide production in male rats. We have previously shown that there is a gender difference in neointimal formation after balloon injury using cerivastatin (data not shown). Therefore, the sexual dimorphic response seems to be a class effect of statins. We have not examined this phenomenon in other animal species; therefore, further studies are needed to conclude whether or not these findings are rat-specific. In our time-window study, we found that short-term (7 days) atorvastatin treatment from day − 3 to day 3 was sufficient to maximally inhibit neointimal formation, and had a 23% inhibition that was comparable to that seen with continuous atorvastatin treatment from day − 3 to day 14. On the other hand, short-term treatment (7 days) with atorvastatin from day 7 to day 14 failed to reduce neointimal thickening in the injured artery. Therefore, atorvastatin reduced neointimal formation by altering cellular and/or molecular events that occur early in response to vascular injury. Similar results have been obtained with the administration of 17β-estradiol to balloon-injured rat carotid arteries (Mori et al., 2000). NADPH-dependent superoxide production of injured carotid arteries was markedly increased at day 2; this increase continued until day 7 and then returned to the level of uninjured vessels at day 14. Therefore, our findings suggest that atorvastatin, as well as estrogen,
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inhibits neointimal formation probably by attenuating the initial rise in NADPH-dependent superoxide production. In summary, we have demonstrated that there is a sexual dimorphic response with atorvastatin treatment in balloon-injured rat carotid arteries, and that NADPH oxidase activity shows a similar sexual dimorphic trend. Thus, the sexual dimorphic response can be at least partially explained by the difference between the genders in the oxidative status due to treatment; these findings seem to be independent of the male sex hormone. Furthermore, we demonstrated that short-term atorvastatin treatment started before vascular injury was effective in preventing neointimal formation. These findings suggest that it is important to suppress NADPH oxidase activation around the time of vascular injury. Our results also suggest that statins may be an alternative therapy for primary and secondary prevention of cardiovascular diseases in postmenopausal women who previously may have been considered hormone replacement therapy candidates. References Barrett-Connor, E., 1997. Sex differences in coronary heart disease: why are women so superior? The 1995 Ancel Keys Lecture. Circulation 95, 252–264. Chen, J., Zhang, Z.G., Li, Y., Wang, Y., Wang, L., Jiang, H., Zhang, C., Lu, M., Katakowski, M., Feldkamp, C.S., Chopp, M., 2003. Statins induce angiogenesis, neurogenesis, and synaptogenesis after stroke. Ann. Neurol. 53, 743–751. Dobrzycka, B., Dobrzycki, S., Lenczewski, A., Terlikowski, S., Kulikowski, M., 2003. Effects of simvastatin only or in combination with continuous combined hormone replacement therapy on lipid levels in hypercholesterolemic women. Ginekol. Pol. 74, 937–942. Florian, M., Freiman, A., Magder, S., 2004. Treatment with 17-beta-estradiol reduces superoxide production in aorta of ovariectomized rats. Steroids 69, 779–787. Grady, D., Rubin, S.M., Petitti, D.B., Fox, C.S., Black, D., Ettinger, B., Ernster, V.L., Cummings, S.R., 1992. Hormone therapy to prevent disease and prolong life in postmenopausal women. Ann. Intern. Med. 117, 1016–1037. Harrison, D., Griendling, K.K., Landmesser, U., Hornig, B., Drexler, H., 2003. Role of oxidative stress in atherosclerosis. Am. J. Cardiol. 91, 7A–11A. Herrington, D.M., et al., for the HERS study group, 2002. Statin therapy, cardiovascular events, and total mortality in the Heart and Estrogen/ Progestin Replacement Study (HERS). Circulation 105, 2962–2967. Hulley, S., Grady, D., Bush, T., Furberg, C., Herrington, D., Riggs, B., Vittinghoff, E., 1998. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA 280, 605–613. Itoh, S., Umemoto, S., Hiromoto, M., Toma, Y., Tomochika, Y., Aoyagi, S., Tanaka, M., Fujii, T., Matsuzaki, M., 2002. Importance of NAD(P)H Oxidase-mediated oxidative stress and contractile type smooth muscle myosin heavy chain SM2 at the early stage of atherosclerosis. Circulation 105, 2288–2295. Jovanovic, S., Jovanovic, A., 1997. Remodelling of guinea-pig aorta during pregnancy: selective alteration of endothelial cells. Hum. Reprod. 12, 2297–2302. Jovanovic, S., Jovanovic, A., 1998. Pregnancy is associated with hypotrophy of carotid artery endothelial and smooth muscle cells. Hum. Reprod. 13, 1074–1078. Koh, K.K., Cardillo, C., Bui, M.N., Hathaway, L., Csako, G., Waclawiw, M.A., Panza, J.A., Cannon III, R.O., 1999. Vascular effects of estrogen and
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Gender difference of atorvastatin's vasoprotective effect in balloon-injured rat carotid arteries Daisuke Kurumazuka a , Tatsuhiko Mori b,⁎, Noriko Matsumoto a , Hisashi Shirakawa a , Sayaka Kimura a , Daisuke Nakano a , Tetsuya Hayashi b , Yasushi Kitaura b , Yasuo Matsumura a b
a Department of Pharmacology, Osaka University of Pharmaceutical Sciences, Japan Department of Medicine III, Osaka Medical College, 2–7 Daigakumachi, Takatsuki, Osaka, 569-8686, Japan
Received 27 June 2006; received in revised form 14 September 2006; accepted 19 September 2006 Available online 27 September 2006
Abstract The effect of estrogen on neointimal formation in injured rat arteries has been reported to be a sexual dimorphic effect. Recently, it has been reported that 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exhibit vasoprotective effects, which are independent of their cholesterol-lowering effects. In this study, we examined the gender differences of atorvastatin's effect on neointimal formation in balloon-injured rat arteries. Male and female Sprague Dawley rats underwent gonadectomy and balloon injury of the carotid artery. Ovariectomized female, as well as intact and castrated male, rats exhibited marked neointimal formation. Treatment with atorvastatin significantly reduced neointimal formation at day 14 (14 days after injury) and NADPH oxidase-dependent superoxide production at day 2 in ovariectomy, but not in intact and castrated males. In ovariectomized rats, 7 days of atorvastatin treatment from days −3 to 3 but not from days 7 to 14 suppressed neointimal formation at day 14. In this study, we showed that atorvastatin's effect on neointimal formation was female-specific and was more marked in ovariectomized female rats. NADPH oxidase-dependent superoxide production may be involved in the mechanism of the sexual dimorphic response seen in response to atorvastatin treatment. Furthermore, the results suggest the importance of treatment in the early phase after vascular injury. © 2006 Elsevier B.V. All rights reserved. Keywords: Gender difference; Statin; NADPH-dependent superoxide production; Vasoprotection
1. Introduction In the past, the cardiovascular protection thought to be offered by hormone replacement therapy (Barrett-Connor, 1997; Grady et al., 1992; Stampfer et al., 1991) for postmenopausal women was primarily attributed to the improvement of the serum lipid profile. However, recent reports have suggested that estrogen has direct protective effects, including increasing nitric oxide production, suppressing adhesion molecule activity, suppressing smooth muscle proliferation and migration, and suppressing superoxide production (Florian et al., 2004; Mendelsohn and Karas, 1999; Miller et al., 2003; Ranki et al., 2002; Tolbert and Oparil, 2001). Nevertheless, the Heart Estrogen–Progestin Replacement Study (HERS) (Hulley et al., ⁎ Corresponding author. Tel.: +81 72 683 1221; fax: +81 72 684 6598. E-mail address: [email protected] (T. Mori). 0014-2999/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2006.09.036
1998) and the Women's Health Initiative Clinical Trial and observational study (WHI) (Rossouw et al., 2002) did not show any benefit of hormone replacement therapy. Furthermore, WHI indicated that the hormone replacement therapy was associated with significant increases in the incidences of breast cancer and thromboembolic diseases (Rossouw et al., 2002). Like estrogen, 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors (statins) exert a vasoprotective effect by improving the serum lipid profile and by anti-inflammatory mechanisms that are independent of their lipid-lowering effect (Schonbeck and Libby, 2004). Increased arterial superoxide production generated by vascular NADPH oxidase has been reported to play a critical role in the pathogenesis of atherosclerosis (Zalba et al., 2005). Both estrogen and statins have been reported to exhibit anti-oxidative effects through suppression of NADPH oxidase (Florian et al., 2004; Wassmann et al., 2001). Recent reports have shown that estrogen has a
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Fig. 3. Intima to media ratios (mean ± S.E.M.) of carotid arteries at 14 days after balloon injury. The intima to media ratios were increased in ovariectomized female compared to intact females, and the ratios were significantly suppressed by atorvastatin. However, in intact and castrated males, the increase in neointimal formation was not suppressed by atorvastatin. Open bar; vehicle treatment, and hatched bar; atorvastatin treatment. ⁎P b 0.05, compared with vehicle in each. #P b 0.05 compared with intact female rats.
Fig. 1. Light micrographs of neointimal formation in balloon-injured carotid arteries of female rats at 14 days after balloon injury. The neointimal formation observed in ovariectomized female + vehicle was more prominent than in intact females. Atorvastatin treatment significantly suppressed neointimal formation.
vasoprotective effects of atorvastatin; 3) short-term treatment with atorvastatin has a long-term treatment effect; and 4) differences in NADPH oxidase activity underlie the gender difference in atorvastatin's effects. 2. Methods
sexual dimorphic vasoprotective pattern and that female rats have a more favorable treatment effect than male rats in the balloon-injured rat carotid artery model (Tolbert and Oparil, 2001). The mechanisms for the gender difference have not been fully elucidated, though a study did show that the estrogeninduced inhibition of the vascular injury response was mediated by molecular events that occur early after injury (Mori et al., 2000). In the present study, we examined whether: 1) atorvastatin treatment suppresses neointimal formation after balloon injury in rat carotid arteries; 2) there is a gender difference in the
2.1. Animal preparation Ten-week old male and female Sprague Dawley rats were obtained from Japan SLC, Inc., (Shizuoka). The experimental protocols and animal care methods were approved by the Experimental Animal Research Committee at Osaka University of Pharmaceutical Sciences. Under anesthesia using an intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg), the rats either had gonadectomy (ovariectomy for female rats and castration for male rats) or sham surgery. A week later, daily oral administration of atorvastatin (3 mg/kg/day in 0.5% sodium Table 1 Effects on myointimal proliferation after balloon injury of carotid arteries of female intact and ovariectomized rats of atorvastatin 3 mg·kg− 1·d− 1 and vehicle 2 mL·kg− 1·d− 1 treatment for 14 days
Fig. 2. Light micrographs of neointimal formation in balloon-injured carotid arteries of male rats at 14 days after balloon injury. Prominent neointimal formation is observed in the intact castrated male. Atorvastatin treatment could not suppress neointimal formation in intact castrated males.
Intact females
Ovariectomized females
Vehicle
(n = 6)
(n = 6)
Body weight, g Area of media, mm2 Area of intima, mm2 Intima to media ratios
218 ± 4 0.131 ± 0.003 0.133 ± 0.007 1.019 ± 0.038b
244 ± 6a 0.127 ± 0.008 0.166 ± 0.018 1.288 ± 0.081a
Atorvastatin
(n = 4)
(n = 8)
Body weight, g Area of media, mm2 Area of intima, mm2 Intima to media ratios
231 ± 2 0.147 ± 0.004 0.146 ± 0.009 0.992 ± 0.052b
253 ± 3a 0.122 ± 0.003 0.123 ± 0.008b 1.000 ± 0.050b
Morphometric analysis of rat carotid arteries 14 days after balloon injury in intact and ovariectomized female rats. Each value represents the mean ± S.E.M. aP b 0.05 compared with intact females with vehicle. b P b 0.05 compared with ovariectomized females with vehicle.
D. Kurumazuka et al. / European Journal of Pharmacology 553 (2006) 263–268 Table 2 Effects on myointimal proliferation after balloon injury of carotid arteries of male intact and castrated rats of atorvastatin 3 mg·kg− 1·d− 1 and vehicle 2 mL·kg− 1·d− 1 treatment for 14 days Intact males
Castrated males
Vehicle
(n = 8)
(n = 8)
Body weight, g Area of media, mm2 Area of intima, mm2 Intima to media ratios
369 ± 4 0.142 ± 0.005 0.159 ± 0.012 1.110 ± 0.040
349 ± 6 0.154 ± 0.006 0.176 ± 0.019 1.126 ± 0.082
Atorvastatin
(n = 5)
(n = 7)
Body weight, g Area of media, mm2 Area of intima, mm2 Intima to media ratios
374 ± 8 0.134 ± 0.007 0.157 ± 0.022 1.116 ± 0.105
348 ± 7 0.149 ± 0.006 0.179 ± 0.011 1.195 ± 0.040
Morphometric analysis of rat carotid arteries 14 days after balloon injury in intact and castrated male rats. Each value represents the mean ± S.E.M.
carboxymethylcellulose (CMC-Na)), supplied by Pfizer, or vehicle (0.5% CMC-Na in water) was started. In most of the rats, the administration of atorvastatin or vehicle was started 3 days before (day − 3) and continued until 14 days after balloon injury (day 14). Furthermore, some of the ovariectomized female rats were divided into 4 groups and treated with atorvastatin or vehicle as follows: 1) atorvastatin from day − 3 through day 14; 2) atorvastatin from day − 3 to day 3; 3) atorvastatin from day 7 to day 14; and 4) vehicle treatment from day − 3 to day 14. Neointimal formation was evaluated at day 14.
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using ovariectomized female rats sacrificed at days 1, 2, 3, 7, and 14. Given the results of this protocol, we then decided to measure the NADPH-dependent superoxide production in all groups 2 days after balloon injury. Both injured (right) and uninjured (left) common carotid arteries were cleared of adherent adipose and loose connective tissue in situ and were harvested in ice-cold modified Krebs–HEPES buffer containing (in mmol/L): NaCl 99.01, HEPES 20, KCl 4.69, MgSO4 0.59, KH2HPO4 1.03, NaHPO4 25, CaCl2 1.41, and glucose 11.1 (pH 7.4). The tissues were then gently flushed with cold buffer to remove blood from the lumen and were cut into 3 segments. The segments were incubated with NADPH (100 μM) in buffer at 37 °C for 15 min s. Lucigeninenhanced chemiluminescence was measured in 2 mL Krebs– HEPES buffer containing lucigenin (5 μmol/L) using a Berthold FB12 single-tube luminometer, modified to maintain a sample temperature of 37 °C. Chemiluminescence was measured continuously for 15 min s after allowing for dark adaptation and was expressed as relative light units per minute per milligram vessel dry weight (RLU/min/mg). 2.5. Statistical analysis All values are expressed as mean ± S.E.M. The data were statistically analyzed using one-way analysis of variance combined with Dunnett's multiple range test for multiple comparisons. Differences were considered significant when the P value was less than 0.05. 3. Results 3.1. Response of right carotid arteries to balloon injury
2.2. Balloon injury procedure Rats were anesthetized using intraperitoneal injection of ketamine (80 mg/kg) and xylazine (5 mg/kg), and the right carotid artery was injured with a 2F Fogarty balloon catheter (Baxter International, Deerfield, IL, USA) (Mori et al., 2000). The left carotid artery was not damaged. 2.3. Morphometric analysis
Ovariectomized female rats showed an increased body weight compared to intact female rats. Castration had no effect on the body weight of male rats. Atorvastatin treatment itself had no effect on the body weight of male and female rats. Neointimal formation was more marked in ovariectomized female rats than in intact female rats. In male rats, significant neointimal formation was also observed, which was not affected by castration. Atorvastatin significantly reduced neointimal formation
Two weeks after balloon injury of the right carotid artery, the rats were sacrificed with a sodium pentobarbital overdose (75 mg/kg) and perfused with 10% formalin to fix the arteries. Morphometric analysis of each arterial segment was performed with a computer-based Motic Image Plus 2.0 Morphometric system (Shimadzu, Kyoto). The degree of neointimal formation of the injured carotid artery was expressed as the absolute area of neointima and the intima to media ratios. 2.4. NADPH oxidase activity NADPH oxidase activity was measured based on the degree of NADPH-dependent superoxide production in the isolated carotid arteries assessed by a lucigenin-enhanced assay as previously described (Pu et al., 2003). At first, we examined the time course of NADPH-dependent superoxide production after balloon injury
Fig. 4. The time course of NADPH-dependent superoxide production after balloon injury using ovariectomized female rats sacrificed at days 1, 2, 3, 7, and 14. NADPH-dependent superoxide production in injured carotid arteries began to increase at day 2; this increase continued through day 7, and then returned to the level of uninjured carotid arteries at day 14. Each value represents the mean ± S.E.M. ⁎P b 0.01, compared with sham.
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Fig. 5. Superoxide production depended on the level of NADPH oxidase in uninjured and injured carotid arteries 2 days after balloon injury. The production in injured carotid arteries was increased more than in uninjured carotid arteries. As well, the increase in injured arteries was more prominent in ovariectomized female rats, male intact rats, and castrated male rats, compared to intact female rats. Atorvastatin treatment suppressed the production in ovariectomized female rats but not in male (intact or castrated) rats. Each value represents the mean ± S.E.M. Open bar; vehicle treatment, and hatched bar; atorvastatin treatment. ⁎P b 0.01, compared with ovariectomized female + vehicle.
in ovariectomized female rats (Fig. 1) but not in male (intact or castrated) rats (Fig. 2). In intact females, neointimal formation was not affected by atorvastatin. Morphometric analysis showed that the neointimal area and the intima to media ratios were attenuated by atorvastatin in ovariectomized female rats, but not in male (intact or castrated) rats. (Fig. 3, Tables 1 and 2). 3.2. NADPH-dependent superoxide production in injured and uninjured carotid arteries NADPH-dependent superoxide production in injured carotid arteries began to increase at day 2; this increase continued through day 7, and then returned to the level of uninjured carotid arteries at day 14 (Fig. 4). NADPH-dependent superoxide production in the uninjured carotid arteries was similar in all groups, and atorvastatin did not affect basal NADPH-dependent superoxide production in uninjured arteries (Fig. 5). In injured carotid arteries, NADPH-dependent superoxide production was increased in all rats, but the increase was less in intact female rats than in the other groups. Treatment with atorvastatin significantly suppressed NADPH-dependent superoxide production in
Fig. 6. Intima to media ratios (mean ± S.E.M.) of balloon-injured carotid arteries of ovariectomized female rats treated with atorvastatin or with vehicle for the time periods indicated. Short-term treatment from day − 3 to day 3 but not from day 7 to day 14 shows similar neointimal suppression to treatment from day −3 to day 14. ⁎P b 0.05 compared with vehicle.
the injured arteries of ovariectomized female rats but not in the arteries of male (intact or castrated) rats (Fig. 5). 3.3. Time-window response of balloon-injured right carotid arteries in rats Neointimal formation was reduced by 26% with atorvastatin treatment from day − 3 to day 14 (Fig. 6). Seven-day treatment with atorvastatin from day − 3 to day 3 also suppressed neointimal formation at day 14, with a 23% inhibition that was almost the same as that seen with day − 3 to day 14 treatment. However, 7-day treatment starting at day 7 did not suppress neointimal formation. 4. Discussion The major findings of the present study are: 1) atorvastatin treatment markedly attenuated neointimal formation in ovariectomized female rats but not in male (intact or castrated) rats; 2) treatment with atorvastatin significantly decreased NADPHdependent superoxide production in ovariectomized female rats but not in male (intact or castrated) rats; and 3) short-term atorvastatin treatment from day −3 to day 3 reduced neointimal formation to a level comparable to that seen with continuous atorvastatin treatment given from 3 days before to 14 days after injury. These results suggest that atorvastatin attenuates neointimal formation in balloon-injured vessels by reducing the production of oxidative stress. Most importantly, atorvastatin's effect was observed only in female rats, suggesting that there may be a marked gender difference in the mechanism of atorvastatin's vasoprotective effect. Gender differences in the vasoprotective effects of statins have not been shown in clinical studies. In these studies, patients with a high cholesterol level were recruited, and the vasoprotective effect of statin treatment was considered to have been the result of the lipid lowering effect rather than any other effects. In our experiment, the dosage of atorvastatin (3 mg/kg/
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day) used had no effect on the lipid profile (Chen et al., 2003). Therefore, the gender difference in atorvastatin's vasoprotective effect in this study was not dependent on the lipid-lowering effect of atorvastatin. Thus, our results appear to differ from the results of clinical studies. However, recently, Seed showed that in the three randomized controlled trials, the Scandinavian Simvastatin Survival Study (4S), the Cholesterol And Recurrent Events study (CARE) and The Long-term Intervention with Pravastatin in Ischaemic Disease (LIPID), women accounted for approximately 20% of the subjects in each study, cardiovascular events were fewer in women on active medication but in no single study were there sufficient numbers to result in a significant reduction in cardiovascular or total mortality, even in the 8-year follow up of the 4S (Seed, 2002). This recent report suggests that there is a gender difference in the effect of statins on cardiovascular events in clinical trials. Interestingly, the gender-dependent effect of atorvastatin is very similar to the vasoprotective effect attributed to estrogen. Previous results (Tolbert and Oparil, 2001) and our present results demonstrate that: 1) gonadectomy of female rats was associated with a more robust neointimal proliferative response to balloon injury; and 2) gonadectomy of male rats did not alter the neointimal response. These results suggest that estrogen is an important factor for decreased neointimal formation in intact female rats. Indeed, it has been shown that estrogen administration to ovariectomized female rats attenuated neointimal formation (Tolbert and Oparil, 2001). Jovanovic showed the pregnancy associated hypotrophy of carotid and thoracic endothelial and smooth muscle cells in guinea pig (Jovanovic and Jovanovic, 1997, 1998). These results suggest that the estrogen effects on arterial remodeling not only in injured but also intact artery. To our knowledge, there are no reports suggesting that there is a gender difference in the metabolism of atorvastatin in rats or that atorvastatin's metabolism is affected by sex hormones to a large extent. Therefore, the apparent lack of effect of atorvastatin in male rats could not be due to a reduced plasma concentration of atorvastatin in male rats. Some studies of postmenopausal women found that statin treatment in addition to hormone replacement therapy had additive cholesterol lowering (Dobrzycka et al., 2003; Koh et al., 1999) and antioxidative effects (Seeger et al., 2000), as well as providing additional cardiovascular risk reduction (Herrington et al., 2002). These effects may be partially the result of the cholesterol lowering effects, but Mueck et al. reported that the estrogen/statin combination had a beneficial effect on biochemical markers of endothelial function in human coronary artery cell cultures (Mueck et al., 2001). Furthermore, statins have been reported to inhibit the proliferation of estrogen receptor-negative human breast cancer cells (Mueck et al., 2003). Therefore, the direct vasoprotective mechanisms of estrogen and statins appear to be somewhat different; the effect of statins is not mediated by the estrogen receptor. In this study, we could not examine the effect of an estrogen receptor blocker combined with statin treatment. Nevertheless, the statin's vasoprotective effect seems to be positioned downstream to the estrogen receptor in this receptor signaling pathway. Oxidative stress plays a crucial role in the pathogenesis of atherosclerotic vascular disease (Harrison
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et al., 2003). Recently, reactive oxygen species have also been implicated in other pathological processes that occur in the vessel wall, including endothelial dysfunction, activation of matrix metalloproteinases, and vascular smooth muscle cell (VSMC) migration, growth, and apoptosis (Taniyama and Griendling, 2003). Increased NADPH oxidase and reactive oxygen species have been reported in coronary arteries after balloon injury (Shi et al., 2001). Overall, oxidative stress appears to play many important roles in the pathogenesis of vascular diseases, including the neointimal formation of injured arteries (Zalba et al., 2005). We also found that: 1) superoxide production generated by vascular NADPH oxidase was increased in injured carotid arteries compared with uninjured carotid arteries; 2) superoxide production in injured arteries was further increased by gonadectomy in female but not in male rats; and 3) treatment with atorvastatin significantly decreased superoxide production in ovariectomized female rats but not in male (intact or castrated) rats. Since superoxide production levels appear to be correlated with the extent of neointimal formation, NADPH oxidase and its product, superoxide, may be involved to a large extent in neointimal formation in injured arteries. Our results also suggest that atorvastatin attenuates superoxide production by NADPH oxidase in injured arteries. Independent of their lipid-lowering effects, statins have been shown to reduce oxidative stress and gene expression of p22phox, an essential subunit of NADPH oxidase, which is present in the vasculature (Itoh et al., 2002; Wassmann et al., 2001). The molecular mechanism underlying atorvastatin's effect is still unclear. Given our present data, estrogen appears to maintain a low level of NADPH-dependent superoxide production in balloon-injured carotid arteries, and when given to ovariectomized rats, atorvastatin reduced the exacerbation of NADPH-dependent superoxide production through its antioxidative properties. In this study, we could not determine why atorvastatin was ineffective in suppressing NADPH-dependent superoxide production in male rats. We have previously shown that there is a gender difference in neointimal formation after balloon injury using cerivastatin (data not shown). Therefore, the sexual dimorphic response seems to be a class effect of statins. We have not examined this phenomenon in other animal species; therefore, further studies are needed to conclude whether or not these findings are rat-specific. In our time-window study, we found that short-term (7 days) atorvastatin treatment from day − 3 to day 3 was sufficient to maximally inhibit neointimal formation, and had a 23% inhibition that was comparable to that seen with continuous atorvastatin treatment from day − 3 to day 14. On the other hand, short-term treatment (7 days) with atorvastatin from day 7 to day 14 failed to reduce neointimal thickening in the injured artery. Therefore, atorvastatin reduced neointimal formation by altering cellular and/or molecular events that occur early in response to vascular injury. Similar results have been obtained with the administration of 17β-estradiol to balloon-injured rat carotid arteries (Mori et al., 2000). NADPH-dependent superoxide production of injured carotid arteries was markedly increased at day 2; this increase continued until day 7 and then returned to the level of uninjured vessels at day 14. Therefore, our findings suggest that atorvastatin, as well as estrogen,
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inhibits neointimal formation probably by attenuating the initial rise in NADPH-dependent superoxide production. In summary, we have demonstrated that there is a sexual dimorphic response with atorvastatin treatment in balloon-injured rat carotid arteries, and that NADPH oxidase activity shows a similar sexual dimorphic trend. Thus, the sexual dimorphic response can be at least partially explained by the difference between the genders in the oxidative status due to treatment; these findings seem to be independent of the male sex hormone. Furthermore, we demonstrated that short-term atorvastatin treatment started before vascular injury was effective in preventing neointimal formation. These findings suggest that it is important to suppress NADPH oxidase activation around the time of vascular injury. Our results also suggest that statins may be an alternative therapy for primary and secondary prevention of cardiovascular diseases in postmenopausal women who previously may have been considered hormone replacement therapy candidates. References Barrett-Connor, E., 1997. Sex differences in coronary heart disease: why are women so superior? The 1995 Ancel Keys Lecture. Circulation 95, 252–264. Chen, J., Zhang, Z.G., Li, Y., Wang, Y., Wang, L., Jiang, H., Zhang, C., Lu, M., Katakowski, M., Feldkamp, C.S., Chopp, M., 2003. Statins induce angiogenesis, neurogenesis, and synaptogenesis after stroke. Ann. Neurol. 53, 743–751. Dobrzycka, B., Dobrzycki, S., Lenczewski, A., Terlikowski, S., Kulikowski, M., 2003. Effects of simvastatin only or in combination with continuous combined hormone replacement therapy on lipid levels in hypercholesterolemic women. Ginekol. Pol. 74, 937–942. Florian, M., Freiman, A., Magder, S., 2004. Treatment with 17-beta-estradiol reduces superoxide production in aorta of ovariectomized rats. Steroids 69, 779–787. Grady, D., Rubin, S.M., Petitti, D.B., Fox, C.S., Black, D., Ettinger, B., Ernster, V.L., Cummings, S.R., 1992. Hormone therapy to prevent disease and prolong life in postmenopausal women. Ann. Intern. Med. 117, 1016–1037. Harrison, D., Griendling, K.K., Landmesser, U., Hornig, B., Drexler, H., 2003. Role of oxidative stress in atherosclerosis. Am. J. Cardiol. 91, 7A–11A. Herrington, D.M., et al., for the HERS study group, 2002. Statin therapy, cardiovascular events, and total mortality in the Heart and Estrogen/ Progestin Replacement Study (HERS). Circulation 105, 2962–2967. Hulley, S., Grady, D., Bush, T., Furberg, C., Herrington, D., Riggs, B., Vittinghoff, E., 1998. Randomized trial of estrogen plus progestin for secondary prevention of coronary heart disease in postmenopausal women. JAMA 280, 605–613. Itoh, S., Umemoto, S., Hiromoto, M., Toma, Y., Tomochika, Y., Aoyagi, S., Tanaka, M., Fujii, T., Matsuzaki, M., 2002. Importance of NAD(P)H Oxidase-mediated oxidative stress and contractile type smooth muscle myosin heavy chain SM2 at the early stage of atherosclerosis. Circulation 105, 2288–2295. Jovanovic, S., Jovanovic, A., 1997. Remodelling of guinea-pig aorta during pregnancy: selective alteration of endothelial cells. Hum. Reprod. 12, 2297–2302. Jovanovic, S., Jovanovic, A., 1998. Pregnancy is associated with hypotrophy of carotid artery endothelial and smooth muscle cells. Hum. Reprod. 13, 1074–1078. Koh, K.K., Cardillo, C., Bui, M.N., Hathaway, L., Csako, G., Waclawiw, M.A., Panza, J.A., Cannon III, R.O., 1999. Vascular effects of estrogen and
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