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Increased asymmetric dimethylarginine and enhanced inflammation are associated with impaired vascular reactivity in women with endometriosis
Atherosclerosis 219 (2011) 784–788
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Increased asymmetric dimethylarginine and enhanced inflammation are associated with impaired vascular reactivity in women with endometriosis Shoko Kinugasa, Koichi Shinohara, Akihiko Wakatsuki ∗ Department of Obstetrics and Gynecology, Aichi Medical University, Nagakute, Aichi 480-1195, Japan
a r t i c l e
i n f o
Article history: Received 18 May 2011 Received in revised form 2 August 2011 Accepted 2 August 2011 Available online 10 August 2011 Keywords: Endometriosis Inflammation Flow-mediated vasodilation Asymmetric dimethylarginine (ADMA) Endothelial dysfunction
a b s t r a c t Objective: Enhanced inflammatory responses which may inhibit vascular reactivity, are associated with endometriosis development. Asymmetric dimethylarginine (ADMA), an inhibitor of endogenous nitric oxide synthase, is also implicated in endothelial dysfunction. We aimed to determine whether plasma ADMA and systemic inflammation are associated with endothelial function in women with endometriosis. Methods: We evaluated 41 women with and 28 women without endometriosis. Plasma levels of lipids and inflammatory markers such as high sensitive-C reactive protein (hs-CRP), serum amyloid protein A (SAA), and interleukin-6 (IL-6) were measured in the two groups. We also measured levels of ADMA and symmetric dimethylarginine (SDMA). High-resolution ultrasonography measured flow-mediated vasodilation (FMD) to assess vasodilatory responses. Results: FMD was significantly lower in women with endometriosis compared to those without endometriosis (8.39 ± 0.43% vs 10.79 ± 0.54%, P = 0.001). While plasma lipid levels did not differ significantly between groups, levels of AMDA, but not SDMA, were significantly higher in women with endometriosis (409.7 ± 10.1 pmol/L vs 383.0 ± 48.3 pmol/L, P = 0.04). Inflammatory markers were also significantly higher in these women (hs-CRP: 1053.3 ± 252.0 ng/mL vs 272.0 ± 83.3 ng/mL, P = 0.02; SAA: 8.00 ± 1.53 g/mL vs 3.82 ± 0.42 g/mL, P = 0.04; IL-6: 2.73 ± 0.75 pg/mL vs 1.05 ± 0.60 pg/mL, P = 0.04). FMD was negatively correlated with plasma levels of ADMA (r = −0.37, P = 0.01) and log hs-CRP (r = −0.34, P = 0.01). Conclusion: Increased plasma ADMA levels and enhanced inflammation are associated with inhibited endothelial function in women with endometriosis. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Endometriosis, a common gynecological disorder characterized by growth of the endometrial gland and stroma outside the uterus, causes several symptoms such as dysmenorrhea, hypermenorrhea, and chronic abdominal pain. Endometriosis has been noted in 20–50% of patients undergoing gynecological laparotomies [1]. Endometriosis is diagnosed in women of reproductive age and is often accompanied by infertility in 5–10% of cases. Endometriosis is accompanied by inflammation, and endometrial tissue can activate macrophages that express scavenger receptors and produce pro-inflammatory cytokines such as interleukin (IL)-6, IL-1, and tumor necrosis factor-alpha. Oxygen free radicals may promote the development of endometriosis and infer-
∗ Corresponding author. Tel.: +81 561 62 3311; fax: +81 561 62 1953. E-mail address: [email protected] (A. Wakatsuki). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.08.005
tility by stimulating the growth and adhesion of endometrial cells in the peritoneal cavity [2]. Endothelial dysfunction is one of the earliest events during the course of atherosclerosis [3]. Nitric oxide (NO), which is produced during arginine oxidation by NO synthase in endothelial cells, has anti-atherosclerotic effects. Endothelium-dependent vasodilation, which is mediated through the release of vasodilators such as NO [4], may be affected by several factors. Vascular inflammation may decrease the production and activity of endothelium-derived NO [5]. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis, also suppresses vascular NO production, while symmetric dimethylarginine (SDMA), a stereoisomer of ADMA, lacks NO synthesis inhibitory activity. Reduced NO activity induces leukocyte adhesion, thrombosis, and vasoconstriction, and accelerates the progression of atherosclerosis [6]. Moreover, plasma ADMA levels are associated with higher levels of C-reactive protein (CRP), an indicator of systemic inflammation [7], and are increased in subjects with atherosclerotic diseases [8].
S. Kinugasa et al. / Atherosclerosis 219 (2011) 784–788
In this study, we measured endothelium-dependent vasodilation in women with and without endometriosis to determine whether vascular reactivity is inhibited in women with endometriosis. We also measured levels of ADMA and SDMA and markers of inflammation and analyzed their associations with endothelial function.
2. Methods 2.1. Subjects We evaluated 41 Japanese women with American Fertility Society (AFS) stage III to IV ovarian endometriomas, and 28 Japanese women without endometriosis between April 1, 2009 and March 31, 2010. Surgeons completed operative records, which noted the presence or absence of endometriosis and the stage of endometriosis according to the revised AFS criteria. Exclusion criteria included the presence of diseases such as diabetes mellitus, hypertension, cardiovascular disease, dyslipidemia, systemic lupus erythematosus, and any other infection. Women who were smokers and/or used any medication were also excluded. Endometriosis was diagnosed by laparoscopy, and control subjects underwent the same surgery for uterine myomas. Endometriosis was confirmed by histopathologic examination. Written informed consent was obtained from each subject before admission to the study. The study design was approved by the Ethics Committee of Aichi Medical University.
2.2. Laboratory analysis Venous blood samples were obtained between 8:00 and 10:00 AM following a 12-h fasting period. Basal body temperature was used to determine menstrual cycle phase, with all women showing a biphasic basal body temperature pattern. Blood samples were drawn at the mid-follicular phase (day 7–10) of the menstrual cycle. Levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL)-cholesterol were measured using enzymatic methods. Levels of high-density lipoprotein (HDL)cholesterol were determined using similar methods after precipitation of apolipoprotein B-containing lipoproteins with sodium phosphotungstate in the presence of magnesium chloride. Levels of estradiol (E2) and follicle-stimulating hormone (FSH) were measured using radioimmunoassays. Plasma levels of CA-125, one of the markers for evaluating the severity of endometriosis, were measured by enzyme immunoassay. High sensitive (hs)CRP levels were measured using the Behring Latex-Enhanced CRP assay on the Behring Nephelometer Analyzer System (Dade, Behring). Serum amyloid A protein (SAA) levels were determined by a latex agglutination turbidimetric immunoassay. Interleukin6 (IL-6) levels were measured by chemiluminescent enzyme immunoassay.
2.3. ADMA and SDMA Plasma ADMA and SDMA levels were measured by highperformance liquid chromatography (HPLC), using precolumn derivatization with o-phthalaldehyde (OPA). Plasma samples and internal standards were extracted and incubated with the OPA reagent (5.4 mg/mL OPA in borate buffer, pH 8.5 containing 0.4% mercaptoethanol). OPA derivatives of ADMA and SDMA were separated on a C6H5 column (Macherey and Nagel) with the fluorescence monitor set at an excitation wavelength of 340 nm and an emission wavelength of 455 nm [9].
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Table 1 Subjects characteristics and concentrations of plasma lipids.
Age (years) BMI (kg/m2 ) Estradiol (pg/mL) FSH (mIU/mL) CA125 (U/mL) Total cholesterol (mg/dL) Triglyceride (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL)
Endometriosis
Control
± ± ± ± ± ± ± ± ±
38.7 21.6 76.4 18.1 28.2 188.9 83.9 62.1 113.4
40.1 19.8 87.5 11.4 91.5 184.1 83.5 66.6 103.4
1.1 0.4 12.1 1.6 12.1 4.7 5.5 1.8 4.1
± ± ± ± ± ± ± ± ±
P 1.0 0.3 9.0 3.6 4.4 4.2 8.7 1.3 3.3
0.2 0.2 0.4 0.8 0.04 0.9 0.4 0.2 0.9
Data are expressed as mean ± SE; BMI, body mass index; FSH, follicle-stimulating hormone; HDL, high-density lipoprotein; LDL, low-density lipoprotein.
2.4. Endothelial function Patients rested in the supine position for 10 min before initiating the examinations. High-resolution Doppler ultrasonography equipment (Sonovista-Color model MEU-1582, Mochida) with a 10-MHz transducer was used to image the right brachial artery, and vasodilatory responses were measured. A nontortuous segment of the brachial artery was scanned longitudinally 4–5 cm above the elbow, where the clearest image could be obtained. When an adequate transducer position was determined, the skin was marked and the arm was kept in a constant position throughout the study. After baseline images of the brachial artery were obtained and arterial flow velocity was determined, a blood pressure cuff encircling the proximal portion of the arm was inflated to 250 mmHg for 5 min, and then rapidly deflated. Increased blood flow after cuff deflation, termed reactive hyperemia, results in flow-mediated vasodilation (FMD) [10]. Flow velocity in the artery was determined again, and 1 min after cuff deflation, the brachial artery was imaged. Blood pressure and heart rate were recorded during the investigation. The diameter of the brachial artery was measured from the anterior to posterior interface between the media and adventitia (“m” line) at the end of diastole, incident with the R wave on a continuously recorded electrocardiogram. Diameters for four cardiac cycles were determined from the images, and these measurements were averaged. All scans were recorded for later analysis. FMD was calculated as the percent increase in arterial diameter during hyperemia and was used as an index of endotheliumdependent vasodilation [10]. Blood flow was calculated by multiplying the time velocity integral of the angle-corrected Doppler flow signals by the heart rate and the mean crosssectional vessel area. Intraobserver and interobserver variability for repeated measurements were 0.03 ± 0.02 and 0.05 ± 0.03 mm, respectively. Variability for FMD performed on 2 separate days was 2.1 ± 0.9%. 2.5. Statistical analysis Data are expressed as mean ± standard error (SE). Differences in subject characteristics, plasma levels of E2, FSH, CA-125, lipids, inflammatory markers, ADMA and SDMA and FMD were analyzed by Student’s unpaired t-test when there was normal distribution or by Mann–Whitney test when the parameters did not exhibit normal distribution. Regression lines were determined by the least squares method. P < 0.05 was considered significant. 3. Results No significant differences between the endometriosis and control groups were found in age, body mass index (BMI), plasma E2 and FSH levels, and levels of TC, TG, LDL-C, and HDL-C. Plasma CA125 was significantly elevated in the endometriosis group (Table 1).
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S. Kinugasa et al. / Atherosclerosis 219 (2011) 784–788
Table 2 Blood pressure, heart rate, brachial artery diameter and blood flow.
Systolic BP (mmHg) Diastolic BP (mmHg) Heart rate (betas/min) Baseline diameter (mm) Baseline flow (mL/min) Hyperemic flow (%)
Endometriosis
Control
± ± ± ± ± ±
111 71 69 3.36 82.7 396.6
113 73 75 3.24 109.8 311.8
2 1 2 0.09 15.0 43.2
± ± ± ± ± ±
P 2 2 1 0.06 5.1 32.9
Table 3 Concentrations of inflammatory markers and asymmetrical and symmetrical dimethylarginine. Endometriosis
0.8 0.9 0.5 0.6 0.7 0.5
Data are expressed as mean ± SE; BP, blood pressure.
hs-CRP (ng/mL) SAA (g/mL) IL-6 (pg/mL) ADMA (pmol/L) SDMA (pmol/L)
1053.3 8.00 2.73 409.7 359.4
± ± ± ± ±
Control
252.0 1.53 0.75 10.1 58.5
272.0 3.82 1.05 383.0 358.3
± ± ± ± ±
P 83.3 0.42 0.60 48.3 58.8
0.02 0.04 0.04 0.04 0.9
Data are expressed as mean ± SE; hs-CRP, high sensitive-C reactive protein; SAA, serum amyloid protein A; IL-6, interleukin-6; ADMA, asymmetrical dimethylarginine; SDMA, symmetrical dimethylarginine.
P = 0.001
Flow-mediated vasodilation (%)
15
icantly higher in the endometriosis group (Table 3). FMD was significantly negatively correlated with levels of ADMA (r = −0.37, P = 0.01) (Fig. 2A) and log hs-CRP (r = −0.34, P = 0.01) (Fig. 2B).
12
4. Discussion 9
6
3
0
Endometriosis
Control
Fig. 1. Flow-mediated vasodilation in control and endometriosis group. Data are expressed as mean ± SE.
Systolic and diastolic BP, heart rate, brachial artery diameter, blood flow, and percent increase in blood flow induced by reactive hyperemia did not significantly differ between the two groups (Table 2). FMD in the endometriosis group was significantly lower compared with the control group (8.39 ± 0.43% vs 10.79 ± 0.54%, P = 0.001) (Fig. 1). Plasma levels of inflammatory markers such as hs-CRP, SAA, and IL-6, and levels of ADMA, but not SDMA, were signif-
A 18
In this study, we measured endothelial function, which is associated with the development of atherosclerosis. NO, an endothelium-derived relaxing factor, is released in response to increased blood flow during reactive hyperemia. Given that several NO synthase inhibitors suppress endothelium-dependent vasodilation [11], FMD appears to represent a vasodilation-dependent effect mediated by endothelium-derived NO. FMD correlates with the severity and extent of coronary atherosclerosis [12]. FMD has provided valuable insights into early atherosclerosis and the potential reversibility of endothelial dysfunction by various strategies. In our study, FMD was lower in women with endometriosis, indicating that vascular reactivity may be impaired in these women. Endothelial function is influenced by several factors. Decreased plasma estrogen levels are accompanied by the inhibition of endothelial function, while estrogen replacement improves endotheliumdependent vasodilation [13]. Hyperlipidemia and hypertension are also associated with impaired endothelial function. Because blood pressure and plasma estrogen and lipid levels did not differ between the two groups, these factors may not affect endothelial function. ADMA, an inhibitor of NO synthesis, suppresses vascular NO production and impairs vascular reactivity, leading to endothelial dysfunction and vasoconstriction. In this study, levels of ADMA,
B
r = - 0.37 P = 0.01
Flow-mediated vasodilation (%)
Flow-mediated vasodilation (%)
r = - 0.34 P = 0.01
16
16 14 12 10 8
14 12 10 8 6
6 4 200
18
300
400 ADMA (pmol/L)
500
600
4
1
2
3
4
Log hs-CRP
Fig. 2. (A) Relationship between flow-mediated vasodilation and plasma asymmetrical dimethylarginine (ADMA) concentrations. Open and closed circles indicate control and endometriosis group, respectively. (B) Relationship between flow-mediated vasodilation and plasma high sensitive-C reactive protein (hs-CRP) concentrations. Open and closed circles indicate control and endometriosis group, respectively.
S. Kinugasa et al. / Atherosclerosis 219 (2011) 784–788
but not SDMA, which lacks NO synthase inhibitory activity, were elevated in women with endometriosis, and were inversely associated with FMD. This indicates that increased plasma ADMA may be associated with impaired endothelial dysfunction in women with endometriosis. Antoniades et al. have demonstrated that ADMA are associated with increased superoxide production and endothelial NO synthase uncoupling [14]. Thus, ADMA not only inhibits NO synthase but also increases superoxide production which may further decrease NO bioavailability. ADMA is metabolized to citrulline by dimethylarginine dimethylaminohydrolase (DDAH), which is present in many tissues. ADMA levels can increase in plasma due to increased production, impaired metabolic degradation, or reduced clearance. Although dimethylarginine is excreted via kidneys and accumulates in subjects with chronic renal failure, our subjects did not show signs of renal failure. Estrogen has been reported to decrease plasma ADMA levels by stimulating DDAH activity [15]. In this study, it is likely that plasma estrogen did not affect ADMA levels because E2 levels did not differ between the two groups. Because plasma ADMA levels positively correlate with CRP levels [7], inflammation may elevate ADMA levels in women with endometriosis. Inflammatory responses may be associated with decreased endothelium-derived NO [16], which promotes leukocyte adhesion and thrombosis formation, thereby leading to atherosclerosis. Inflammation activates macrophages that induce IL-6 secretion. This increase in IL-6 secretion stimulates hepatic CRP production. Previous studies have reported that CRP levels negatively correlate with endothelial function [17,18], while other studies have shown no such association between these two parameters [19,20]. Despite this discrepancy, Vita et al. reported that systemic inflammation may contribute to impaired vasomotor function in microvessels in the Framingham Offspring Study, which involved a large number of subjects [21]. Since SAA is synthesized in the liver in response to infection and inflammation like CRP, it is also recognized as a sensitive marker of inflammation. In the present study, inflammatory markers such as hs-CRP, SAA, and IL-6 were all elevated in women with endometriosis, suggesting an enhanced inflammatory response in these women. In addition, levels of hs-CRP correlated negatively with FMD. This indicates that increased CRP production may be associated with impaired vascular reactivity in women with endometriosis. ´ Szczepanska et al. demonstrated that women with endometriosis have lower levels of scavenging enzymes such as superoxide dismutase and glutathione peroxidase in peritoneal fluid [22]. Oxidative stress has been reported to increase in women with endometriosis [23]. Oxidative stress, particularly the oxidation of LDL, is also a potent inhibitor of endothelium-dependent vascular relaxation [24]. Oxidation of LDL results in the interruption of G protein-dependent stimulation of NO release, and lipid peroxidation products directly block the physiologic action of NO [25]. Because macrophages and neutrophils that generate reactive oxygen species are activated in women with endometriosis, it is likely that enhanced oxidative stress may affect adversely endothelial function in these women. According to Ho et al., FMD was increased in hyperthyroidism patients, while it decreased by the administration of anti-thyroid agents [26]. Gazdag et al. also showed that the addition of levothyoxine increased FMD and plasma CRP levels in patients with thyroidectomy [27]. In addition, thyroid hormone may also influence plasma LDL cholesterol levels. Although we did not measure the plasma levels of thyroid hormone in the present study, plasma LDL cholesterol levels did not differ between the two groups. Accordingly, it is unlikely that thyroid function affected endothelial function and plasma levels of inflammatory markers. As in the case of endometriosis, macrophages are also activated in atherosclerotic tissues in subjects with cardiovascular disease. Chronic inflammatory diseases such as chlamydia pneumoniae and
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dental infection have been reported to increase the risk of coronary heart disease [28,29]. Oxidative stress can also actively contribute to the development of atherosclerotic lesions. Moreover, elevated ADMA is observed in subjects with renal failure, cardiovascular disease, and diabetes mellitus. Based on these findings, women with endometriosis may be at increased risk for myocardial infarction or stroke. Pretta et al. demonstrated that intima–media thickness and distensibility coefficient of the common carotid artery did not differ significantly between women with and without endometriosis, and concluded that women with endometriosis may not be at increased risk for subclinical atherosclerosis [30]. Although women become more susceptible to cardiovascular disease after menopause given their low estrogen levels, subjects in their study were premenopausal women. Thus, postmenopausal women with a previous history of endometriosis may present a different picture. In this study, we found that increased plasma ADMA levels and enhanced inflammation may be associated with endothelial dysfunction. Since endometriosis is an estrogen-dependent disease, chronic inflammation may result in the development of atherosclerosis after menopause. Further studies will be needed to determine whether women with endometriosis are at increased risk for cardiovascular disease.
References [1] Matorras R, Rodíquez F, Pijoan JI, et al. Epidemiology of endometriosis in infertile women. Fertil Steril 1995;63:34–8. [2] Alpay Z, Saed GM, Diamond MP. Female infertility and free radicals: potential role in adhesions and endometriosis. J Soc Gynecol Investig 2006;13:390–8. [3] Gilligan DM, Quyyumi AA, Cannon 3rd RO. Effects of physiological levels of estrogen on coronary vasomotor function in postmenopausal women. Circulation 1994;89:2545–51. [4] Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524–6. [5] Verma S, Wang CH, Li SH, et al. A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis. Circulation 2002;106(8):913–9. [6] Vita JA, Keaney Jr JF. Endothelial function: a barometer for cardiovascular risk? Circulation 2002;106:640–2. [7] Xia W, Feng W, Guan M, et al. Increased levels of asymmetric dimethylarginine and C-reactive protein are associated with impaired vascular reactivity in essential hypertension. Clin Exp Hypertens 2010;32:43–8. [8] Böger RH. The emerging role of asymmetric dimethylarginine as a novel cardiovascular risk factor. Cardiovasc Res 2003;59:824–33. [9] Kielstein JT, Bode-Böger SM, Frölich JC, et al. Asymmetric dimethylarginine, blood pressure, and renal perfusion in elderly subjects. Circulation 2003;107:1891–5. [10] Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endotheliumderived relaxing factor. Am J Physiol 1986;250:H1145–9. [11] Joannides R, Haefeli WE, Linder L, et al. Nitric oxide is responsible for flowdependent dilatation of human peripheral conduit arteries in vivo. Circulation 1995;91:1314–9. [12] Neunteufl T, Katzenschlager R, Hassan A, et al. Systemic endothelial dysfunction is related to the extent and severity of coronary artery disease. Atherosclerosis 1997;129:111–8. [13] Lieberman EH, Gerhard MD, Uehata A, et al. Estrogen improves endotheliumdependent, flow-mediated vasodilation in postmenopausal women. Ann Intern Med 1994;121:936–41. [14] Antoniades C, Shirodaria C, Leeson P, et al. Association of plasma asymmetrical dimethylarginine (ADMA) with elevated vascular superoxide production and endothelial nitric oxide synthase uncoupling: implications for endothelial function in human atherosclerosis. Eur Heart J 2009;30:1142–50. [15] Holden DP, Cartwright JE, Nussey SS, et al. Estrogen stimulates dimethylarginine dimethylaminohydrolase activity and the metabolism of asymmetric dimethylarginine. Circulation 2003;108:1575–80. [16] Widlansky ME, Gokce N, Keaney Jr JF, et al. The clinical implications of endothelial dysfunction. J Am Coll Cardiol 2003;42:1149–60. [17] Tan KC, Chow WS, Tam SC, et al. Atorvastatin lowers C-reactive protein and improves endothelium-dependent vasodilation in type 2 diabetes mellitus. J Clin Endocrinol Metab 2002;87:563–8. [18] Brevetti G, Silvestro A, Di Giacomo S, et al. Endothelial dysfunction in peripheral arterial disease is related to increase in plasma markers of inflammation and severity of peripheral circulatory impairment but not to classic risk factors and atherosclerotic burden. J Vasc Surg 2003;38:374–9. [19] Prasad A, Zhu J, Halcox JP, et al. Predisposition to atherosclerosis by infections: role of endothelial dysfunction. Circulation 2002;106:184–90.
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[20] van Haelst PL, van Doormaal JJ, Asselbergs FW, et al. Correlates of endothelial function and their relationship with inflammation in patients with familial hypercholesterolaemia. Clin Sci 2003;104:627–32. [21] Vita JA, Keaney Jr JF, Larson MG, et al. Brachial artery vasodilator function and systemic inflammation in the Framingham Offspring Study. Circulation 2004;110:3604–9. ´ ´ [22] Szczepanska M, Kozlik J, Skrzypczak J, et al. Oxidative stress may be a piece in the endometriosis puzzle. Fertil Steril 2003;79:1288–93. [23] Shanti A, Santanam N, Morales AJ, et al. Autoantibodies to markers of oxidative stress are elevated in women with endometriosis. Fertil Steril 1999;71:1115–8. [24] Tanner FC, Noll G, Boulanger CM, et al. Oxidized low density lipoproteins inhibit relaxations of porcine coronary arteries. Role of scavenger receptor and endothelium-derived nitric oxide. Circulation 1991;83:2012–20. [25] Chin JH, Azhar S, Hoffman BB. Inactivation of endothelial derived relaxing factor by oxidized lipoproteins. J Clin Invest 1992;89:10–8. [26] Ho WJ, Chen ST, Tsay PK, et al. Enhancement of endothelium-dependent flowmediated vasodilation in hyperthyroidism. Clin Endocrinol 2007;67:505–11. [27] Gazdag A, Nagy EV, Burman KD, et al. Improved endothelial function and lipid profile compensate for impaired hemostatic and inflammatory status in iatro-
genic chronic subclinical hyperthyroidism of thyroid cancer patients on L-t4 therapy. Exp Clin Endocrinol Diabetes 2010;118:381–7. [28] Danesh J, Collins R, Peto R. Chronic infections and coronary heart disease: is there a link? Lancet 1997;350:430–6. [29] Mattila KJ, Valtonen VV, Nieminen MS, et al. Role of infection as a risk factor for atherosclerosis, myocardial infarction, and stroke. Clin Infect Dis 1998;26:719–34. [30] Pretta S, Remorgida V, Abbamonte LH, et al. Atherosclerosis in women with endometriosis. Eur J Obstet Gynecol Reprod Biol 2007;132:226–31.
Contents lists available at ScienceDirect
Atherosclerosis journal homepage: www.elsevier.com/locate/atherosclerosis
Increased asymmetric dimethylarginine and enhanced inflammation are associated with impaired vascular reactivity in women with endometriosis Shoko Kinugasa, Koichi Shinohara, Akihiko Wakatsuki ∗ Department of Obstetrics and Gynecology, Aichi Medical University, Nagakute, Aichi 480-1195, Japan
a r t i c l e
i n f o
Article history: Received 18 May 2011 Received in revised form 2 August 2011 Accepted 2 August 2011 Available online 10 August 2011 Keywords: Endometriosis Inflammation Flow-mediated vasodilation Asymmetric dimethylarginine (ADMA) Endothelial dysfunction
a b s t r a c t Objective: Enhanced inflammatory responses which may inhibit vascular reactivity, are associated with endometriosis development. Asymmetric dimethylarginine (ADMA), an inhibitor of endogenous nitric oxide synthase, is also implicated in endothelial dysfunction. We aimed to determine whether plasma ADMA and systemic inflammation are associated with endothelial function in women with endometriosis. Methods: We evaluated 41 women with and 28 women without endometriosis. Plasma levels of lipids and inflammatory markers such as high sensitive-C reactive protein (hs-CRP), serum amyloid protein A (SAA), and interleukin-6 (IL-6) were measured in the two groups. We also measured levels of ADMA and symmetric dimethylarginine (SDMA). High-resolution ultrasonography measured flow-mediated vasodilation (FMD) to assess vasodilatory responses. Results: FMD was significantly lower in women with endometriosis compared to those without endometriosis (8.39 ± 0.43% vs 10.79 ± 0.54%, P = 0.001). While plasma lipid levels did not differ significantly between groups, levels of AMDA, but not SDMA, were significantly higher in women with endometriosis (409.7 ± 10.1 pmol/L vs 383.0 ± 48.3 pmol/L, P = 0.04). Inflammatory markers were also significantly higher in these women (hs-CRP: 1053.3 ± 252.0 ng/mL vs 272.0 ± 83.3 ng/mL, P = 0.02; SAA: 8.00 ± 1.53 g/mL vs 3.82 ± 0.42 g/mL, P = 0.04; IL-6: 2.73 ± 0.75 pg/mL vs 1.05 ± 0.60 pg/mL, P = 0.04). FMD was negatively correlated with plasma levels of ADMA (r = −0.37, P = 0.01) and log hs-CRP (r = −0.34, P = 0.01). Conclusion: Increased plasma ADMA levels and enhanced inflammation are associated with inhibited endothelial function in women with endometriosis. © 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Endometriosis, a common gynecological disorder characterized by growth of the endometrial gland and stroma outside the uterus, causes several symptoms such as dysmenorrhea, hypermenorrhea, and chronic abdominal pain. Endometriosis has been noted in 20–50% of patients undergoing gynecological laparotomies [1]. Endometriosis is diagnosed in women of reproductive age and is often accompanied by infertility in 5–10% of cases. Endometriosis is accompanied by inflammation, and endometrial tissue can activate macrophages that express scavenger receptors and produce pro-inflammatory cytokines such as interleukin (IL)-6, IL-1, and tumor necrosis factor-alpha. Oxygen free radicals may promote the development of endometriosis and infer-
∗ Corresponding author. Tel.: +81 561 62 3311; fax: +81 561 62 1953. E-mail address: [email protected] (A. Wakatsuki). 0021-9150/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2011.08.005
tility by stimulating the growth and adhesion of endometrial cells in the peritoneal cavity [2]. Endothelial dysfunction is one of the earliest events during the course of atherosclerosis [3]. Nitric oxide (NO), which is produced during arginine oxidation by NO synthase in endothelial cells, has anti-atherosclerotic effects. Endothelium-dependent vasodilation, which is mediated through the release of vasodilators such as NO [4], may be affected by several factors. Vascular inflammation may decrease the production and activity of endothelium-derived NO [5]. Asymmetric dimethylarginine (ADMA), an endogenous inhibitor of NO synthesis, also suppresses vascular NO production, while symmetric dimethylarginine (SDMA), a stereoisomer of ADMA, lacks NO synthesis inhibitory activity. Reduced NO activity induces leukocyte adhesion, thrombosis, and vasoconstriction, and accelerates the progression of atherosclerosis [6]. Moreover, plasma ADMA levels are associated with higher levels of C-reactive protein (CRP), an indicator of systemic inflammation [7], and are increased in subjects with atherosclerotic diseases [8].
S. Kinugasa et al. / Atherosclerosis 219 (2011) 784–788
In this study, we measured endothelium-dependent vasodilation in women with and without endometriosis to determine whether vascular reactivity is inhibited in women with endometriosis. We also measured levels of ADMA and SDMA and markers of inflammation and analyzed their associations with endothelial function.
2. Methods 2.1. Subjects We evaluated 41 Japanese women with American Fertility Society (AFS) stage III to IV ovarian endometriomas, and 28 Japanese women without endometriosis between April 1, 2009 and March 31, 2010. Surgeons completed operative records, which noted the presence or absence of endometriosis and the stage of endometriosis according to the revised AFS criteria. Exclusion criteria included the presence of diseases such as diabetes mellitus, hypertension, cardiovascular disease, dyslipidemia, systemic lupus erythematosus, and any other infection. Women who were smokers and/or used any medication were also excluded. Endometriosis was diagnosed by laparoscopy, and control subjects underwent the same surgery for uterine myomas. Endometriosis was confirmed by histopathologic examination. Written informed consent was obtained from each subject before admission to the study. The study design was approved by the Ethics Committee of Aichi Medical University.
2.2. Laboratory analysis Venous blood samples were obtained between 8:00 and 10:00 AM following a 12-h fasting period. Basal body temperature was used to determine menstrual cycle phase, with all women showing a biphasic basal body temperature pattern. Blood samples were drawn at the mid-follicular phase (day 7–10) of the menstrual cycle. Levels of total cholesterol (TC), triglycerides (TG), and low-density lipoprotein (LDL)-cholesterol were measured using enzymatic methods. Levels of high-density lipoprotein (HDL)cholesterol were determined using similar methods after precipitation of apolipoprotein B-containing lipoproteins with sodium phosphotungstate in the presence of magnesium chloride. Levels of estradiol (E2) and follicle-stimulating hormone (FSH) were measured using radioimmunoassays. Plasma levels of CA-125, one of the markers for evaluating the severity of endometriosis, were measured by enzyme immunoassay. High sensitive (hs)CRP levels were measured using the Behring Latex-Enhanced CRP assay on the Behring Nephelometer Analyzer System (Dade, Behring). Serum amyloid A protein (SAA) levels were determined by a latex agglutination turbidimetric immunoassay. Interleukin6 (IL-6) levels were measured by chemiluminescent enzyme immunoassay.
2.3. ADMA and SDMA Plasma ADMA and SDMA levels were measured by highperformance liquid chromatography (HPLC), using precolumn derivatization with o-phthalaldehyde (OPA). Plasma samples and internal standards were extracted and incubated with the OPA reagent (5.4 mg/mL OPA in borate buffer, pH 8.5 containing 0.4% mercaptoethanol). OPA derivatives of ADMA and SDMA were separated on a C6H5 column (Macherey and Nagel) with the fluorescence monitor set at an excitation wavelength of 340 nm and an emission wavelength of 455 nm [9].
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Table 1 Subjects characteristics and concentrations of plasma lipids.
Age (years) BMI (kg/m2 ) Estradiol (pg/mL) FSH (mIU/mL) CA125 (U/mL) Total cholesterol (mg/dL) Triglyceride (mg/dL) HDL cholesterol (mg/dL) LDL cholesterol (mg/dL)
Endometriosis
Control
± ± ± ± ± ± ± ± ±
38.7 21.6 76.4 18.1 28.2 188.9 83.9 62.1 113.4
40.1 19.8 87.5 11.4 91.5 184.1 83.5 66.6 103.4
1.1 0.4 12.1 1.6 12.1 4.7 5.5 1.8 4.1
± ± ± ± ± ± ± ± ±
P 1.0 0.3 9.0 3.6 4.4 4.2 8.7 1.3 3.3
0.2 0.2 0.4 0.8 0.04 0.9 0.4 0.2 0.9
Data are expressed as mean ± SE; BMI, body mass index; FSH, follicle-stimulating hormone; HDL, high-density lipoprotein; LDL, low-density lipoprotein.
2.4. Endothelial function Patients rested in the supine position for 10 min before initiating the examinations. High-resolution Doppler ultrasonography equipment (Sonovista-Color model MEU-1582, Mochida) with a 10-MHz transducer was used to image the right brachial artery, and vasodilatory responses were measured. A nontortuous segment of the brachial artery was scanned longitudinally 4–5 cm above the elbow, where the clearest image could be obtained. When an adequate transducer position was determined, the skin was marked and the arm was kept in a constant position throughout the study. After baseline images of the brachial artery were obtained and arterial flow velocity was determined, a blood pressure cuff encircling the proximal portion of the arm was inflated to 250 mmHg for 5 min, and then rapidly deflated. Increased blood flow after cuff deflation, termed reactive hyperemia, results in flow-mediated vasodilation (FMD) [10]. Flow velocity in the artery was determined again, and 1 min after cuff deflation, the brachial artery was imaged. Blood pressure and heart rate were recorded during the investigation. The diameter of the brachial artery was measured from the anterior to posterior interface between the media and adventitia (“m” line) at the end of diastole, incident with the R wave on a continuously recorded electrocardiogram. Diameters for four cardiac cycles were determined from the images, and these measurements were averaged. All scans were recorded for later analysis. FMD was calculated as the percent increase in arterial diameter during hyperemia and was used as an index of endotheliumdependent vasodilation [10]. Blood flow was calculated by multiplying the time velocity integral of the angle-corrected Doppler flow signals by the heart rate and the mean crosssectional vessel area. Intraobserver and interobserver variability for repeated measurements were 0.03 ± 0.02 and 0.05 ± 0.03 mm, respectively. Variability for FMD performed on 2 separate days was 2.1 ± 0.9%. 2.5. Statistical analysis Data are expressed as mean ± standard error (SE). Differences in subject characteristics, plasma levels of E2, FSH, CA-125, lipids, inflammatory markers, ADMA and SDMA and FMD were analyzed by Student’s unpaired t-test when there was normal distribution or by Mann–Whitney test when the parameters did not exhibit normal distribution. Regression lines were determined by the least squares method. P < 0.05 was considered significant. 3. Results No significant differences between the endometriosis and control groups were found in age, body mass index (BMI), plasma E2 and FSH levels, and levels of TC, TG, LDL-C, and HDL-C. Plasma CA125 was significantly elevated in the endometriosis group (Table 1).
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Table 2 Blood pressure, heart rate, brachial artery diameter and blood flow.
Systolic BP (mmHg) Diastolic BP (mmHg) Heart rate (betas/min) Baseline diameter (mm) Baseline flow (mL/min) Hyperemic flow (%)
Endometriosis
Control
± ± ± ± ± ±
111 71 69 3.36 82.7 396.6
113 73 75 3.24 109.8 311.8
2 1 2 0.09 15.0 43.2
± ± ± ± ± ±
P 2 2 1 0.06 5.1 32.9
Table 3 Concentrations of inflammatory markers and asymmetrical and symmetrical dimethylarginine. Endometriosis
0.8 0.9 0.5 0.6 0.7 0.5
Data are expressed as mean ± SE; BP, blood pressure.
hs-CRP (ng/mL) SAA (g/mL) IL-6 (pg/mL) ADMA (pmol/L) SDMA (pmol/L)
1053.3 8.00 2.73 409.7 359.4
± ± ± ± ±
Control
252.0 1.53 0.75 10.1 58.5
272.0 3.82 1.05 383.0 358.3
± ± ± ± ±
P 83.3 0.42 0.60 48.3 58.8
0.02 0.04 0.04 0.04 0.9
Data are expressed as mean ± SE; hs-CRP, high sensitive-C reactive protein; SAA, serum amyloid protein A; IL-6, interleukin-6; ADMA, asymmetrical dimethylarginine; SDMA, symmetrical dimethylarginine.
P = 0.001
Flow-mediated vasodilation (%)
15
icantly higher in the endometriosis group (Table 3). FMD was significantly negatively correlated with levels of ADMA (r = −0.37, P = 0.01) (Fig. 2A) and log hs-CRP (r = −0.34, P = 0.01) (Fig. 2B).
12
4. Discussion 9
6
3
0
Endometriosis
Control
Fig. 1. Flow-mediated vasodilation in control and endometriosis group. Data are expressed as mean ± SE.
Systolic and diastolic BP, heart rate, brachial artery diameter, blood flow, and percent increase in blood flow induced by reactive hyperemia did not significantly differ between the two groups (Table 2). FMD in the endometriosis group was significantly lower compared with the control group (8.39 ± 0.43% vs 10.79 ± 0.54%, P = 0.001) (Fig. 1). Plasma levels of inflammatory markers such as hs-CRP, SAA, and IL-6, and levels of ADMA, but not SDMA, were signif-
A 18
In this study, we measured endothelial function, which is associated with the development of atherosclerosis. NO, an endothelium-derived relaxing factor, is released in response to increased blood flow during reactive hyperemia. Given that several NO synthase inhibitors suppress endothelium-dependent vasodilation [11], FMD appears to represent a vasodilation-dependent effect mediated by endothelium-derived NO. FMD correlates with the severity and extent of coronary atherosclerosis [12]. FMD has provided valuable insights into early atherosclerosis and the potential reversibility of endothelial dysfunction by various strategies. In our study, FMD was lower in women with endometriosis, indicating that vascular reactivity may be impaired in these women. Endothelial function is influenced by several factors. Decreased plasma estrogen levels are accompanied by the inhibition of endothelial function, while estrogen replacement improves endotheliumdependent vasodilation [13]. Hyperlipidemia and hypertension are also associated with impaired endothelial function. Because blood pressure and plasma estrogen and lipid levels did not differ between the two groups, these factors may not affect endothelial function. ADMA, an inhibitor of NO synthesis, suppresses vascular NO production and impairs vascular reactivity, leading to endothelial dysfunction and vasoconstriction. In this study, levels of ADMA,
B
r = - 0.37 P = 0.01
Flow-mediated vasodilation (%)
Flow-mediated vasodilation (%)
r = - 0.34 P = 0.01
16
16 14 12 10 8
14 12 10 8 6
6 4 200
18
300
400 ADMA (pmol/L)
500
600
4
1
2
3
4
Log hs-CRP
Fig. 2. (A) Relationship between flow-mediated vasodilation and plasma asymmetrical dimethylarginine (ADMA) concentrations. Open and closed circles indicate control and endometriosis group, respectively. (B) Relationship between flow-mediated vasodilation and plasma high sensitive-C reactive protein (hs-CRP) concentrations. Open and closed circles indicate control and endometriosis group, respectively.
S. Kinugasa et al. / Atherosclerosis 219 (2011) 784–788
but not SDMA, which lacks NO synthase inhibitory activity, were elevated in women with endometriosis, and were inversely associated with FMD. This indicates that increased plasma ADMA may be associated with impaired endothelial dysfunction in women with endometriosis. Antoniades et al. have demonstrated that ADMA are associated with increased superoxide production and endothelial NO synthase uncoupling [14]. Thus, ADMA not only inhibits NO synthase but also increases superoxide production which may further decrease NO bioavailability. ADMA is metabolized to citrulline by dimethylarginine dimethylaminohydrolase (DDAH), which is present in many tissues. ADMA levels can increase in plasma due to increased production, impaired metabolic degradation, or reduced clearance. Although dimethylarginine is excreted via kidneys and accumulates in subjects with chronic renal failure, our subjects did not show signs of renal failure. Estrogen has been reported to decrease plasma ADMA levels by stimulating DDAH activity [15]. In this study, it is likely that plasma estrogen did not affect ADMA levels because E2 levels did not differ between the two groups. Because plasma ADMA levels positively correlate with CRP levels [7], inflammation may elevate ADMA levels in women with endometriosis. Inflammatory responses may be associated with decreased endothelium-derived NO [16], which promotes leukocyte adhesion and thrombosis formation, thereby leading to atherosclerosis. Inflammation activates macrophages that induce IL-6 secretion. This increase in IL-6 secretion stimulates hepatic CRP production. Previous studies have reported that CRP levels negatively correlate with endothelial function [17,18], while other studies have shown no such association between these two parameters [19,20]. Despite this discrepancy, Vita et al. reported that systemic inflammation may contribute to impaired vasomotor function in microvessels in the Framingham Offspring Study, which involved a large number of subjects [21]. Since SAA is synthesized in the liver in response to infection and inflammation like CRP, it is also recognized as a sensitive marker of inflammation. In the present study, inflammatory markers such as hs-CRP, SAA, and IL-6 were all elevated in women with endometriosis, suggesting an enhanced inflammatory response in these women. In addition, levels of hs-CRP correlated negatively with FMD. This indicates that increased CRP production may be associated with impaired vascular reactivity in women with endometriosis. ´ Szczepanska et al. demonstrated that women with endometriosis have lower levels of scavenging enzymes such as superoxide dismutase and glutathione peroxidase in peritoneal fluid [22]. Oxidative stress has been reported to increase in women with endometriosis [23]. Oxidative stress, particularly the oxidation of LDL, is also a potent inhibitor of endothelium-dependent vascular relaxation [24]. Oxidation of LDL results in the interruption of G protein-dependent stimulation of NO release, and lipid peroxidation products directly block the physiologic action of NO [25]. Because macrophages and neutrophils that generate reactive oxygen species are activated in women with endometriosis, it is likely that enhanced oxidative stress may affect adversely endothelial function in these women. According to Ho et al., FMD was increased in hyperthyroidism patients, while it decreased by the administration of anti-thyroid agents [26]. Gazdag et al. also showed that the addition of levothyoxine increased FMD and plasma CRP levels in patients with thyroidectomy [27]. In addition, thyroid hormone may also influence plasma LDL cholesterol levels. Although we did not measure the plasma levels of thyroid hormone in the present study, plasma LDL cholesterol levels did not differ between the two groups. Accordingly, it is unlikely that thyroid function affected endothelial function and plasma levels of inflammatory markers. As in the case of endometriosis, macrophages are also activated in atherosclerotic tissues in subjects with cardiovascular disease. Chronic inflammatory diseases such as chlamydia pneumoniae and
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dental infection have been reported to increase the risk of coronary heart disease [28,29]. Oxidative stress can also actively contribute to the development of atherosclerotic lesions. Moreover, elevated ADMA is observed in subjects with renal failure, cardiovascular disease, and diabetes mellitus. Based on these findings, women with endometriosis may be at increased risk for myocardial infarction or stroke. Pretta et al. demonstrated that intima–media thickness and distensibility coefficient of the common carotid artery did not differ significantly between women with and without endometriosis, and concluded that women with endometriosis may not be at increased risk for subclinical atherosclerosis [30]. Although women become more susceptible to cardiovascular disease after menopause given their low estrogen levels, subjects in their study were premenopausal women. Thus, postmenopausal women with a previous history of endometriosis may present a different picture. In this study, we found that increased plasma ADMA levels and enhanced inflammation may be associated with endothelial dysfunction. Since endometriosis is an estrogen-dependent disease, chronic inflammation may result in the development of atherosclerosis after menopause. Further studies will be needed to determine whether women with endometriosis are at increased risk for cardiovascular disease.
References [1] Matorras R, Rodíquez F, Pijoan JI, et al. Epidemiology of endometriosis in infertile women. Fertil Steril 1995;63:34–8. [2] Alpay Z, Saed GM, Diamond MP. Female infertility and free radicals: potential role in adhesions and endometriosis. J Soc Gynecol Investig 2006;13:390–8. [3] Gilligan DM, Quyyumi AA, Cannon 3rd RO. Effects of physiological levels of estrogen on coronary vasomotor function in postmenopausal women. Circulation 1994;89:2545–51. [4] Palmer RM, Ferrige AG, Moncada S. Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature 1987;327:524–6. [5] Verma S, Wang CH, Li SH, et al. A self-fulfilling prophecy: C-reactive protein attenuates nitric oxide production and inhibits angiogenesis. Circulation 2002;106(8):913–9. [6] Vita JA, Keaney Jr JF. Endothelial function: a barometer for cardiovascular risk? Circulation 2002;106:640–2. [7] Xia W, Feng W, Guan M, et al. Increased levels of asymmetric dimethylarginine and C-reactive protein are associated with impaired vascular reactivity in essential hypertension. Clin Exp Hypertens 2010;32:43–8. [8] Böger RH. The emerging role of asymmetric dimethylarginine as a novel cardiovascular risk factor. Cardiovasc Res 2003;59:824–33. [9] Kielstein JT, Bode-Böger SM, Frölich JC, et al. Asymmetric dimethylarginine, blood pressure, and renal perfusion in elderly subjects. Circulation 2003;107:1891–5. [10] Rubanyi GM, Romero JC, Vanhoutte PM. Flow-induced release of endotheliumderived relaxing factor. Am J Physiol 1986;250:H1145–9. [11] Joannides R, Haefeli WE, Linder L, et al. Nitric oxide is responsible for flowdependent dilatation of human peripheral conduit arteries in vivo. Circulation 1995;91:1314–9. [12] Neunteufl T, Katzenschlager R, Hassan A, et al. Systemic endothelial dysfunction is related to the extent and severity of coronary artery disease. Atherosclerosis 1997;129:111–8. [13] Lieberman EH, Gerhard MD, Uehata A, et al. Estrogen improves endotheliumdependent, flow-mediated vasodilation in postmenopausal women. Ann Intern Med 1994;121:936–41. [14] Antoniades C, Shirodaria C, Leeson P, et al. Association of plasma asymmetrical dimethylarginine (ADMA) with elevated vascular superoxide production and endothelial nitric oxide synthase uncoupling: implications for endothelial function in human atherosclerosis. Eur Heart J 2009;30:1142–50. [15] Holden DP, Cartwright JE, Nussey SS, et al. Estrogen stimulates dimethylarginine dimethylaminohydrolase activity and the metabolism of asymmetric dimethylarginine. Circulation 2003;108:1575–80. [16] Widlansky ME, Gokce N, Keaney Jr JF, et al. The clinical implications of endothelial dysfunction. J Am Coll Cardiol 2003;42:1149–60. [17] Tan KC, Chow WS, Tam SC, et al. Atorvastatin lowers C-reactive protein and improves endothelium-dependent vasodilation in type 2 diabetes mellitus. J Clin Endocrinol Metab 2002;87:563–8. [18] Brevetti G, Silvestro A, Di Giacomo S, et al. Endothelial dysfunction in peripheral arterial disease is related to increase in plasma markers of inflammation and severity of peripheral circulatory impairment but not to classic risk factors and atherosclerotic burden. J Vasc Surg 2003;38:374–9. [19] Prasad A, Zhu J, Halcox JP, et al. Predisposition to atherosclerosis by infections: role of endothelial dysfunction. Circulation 2002;106:184–90.
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S. Kinugasa et al. / Atherosclerosis 219 (2011) 784–788
[20] van Haelst PL, van Doormaal JJ, Asselbergs FW, et al. Correlates of endothelial function and their relationship with inflammation in patients with familial hypercholesterolaemia. Clin Sci 2003;104:627–32. [21] Vita JA, Keaney Jr JF, Larson MG, et al. Brachial artery vasodilator function and systemic inflammation in the Framingham Offspring Study. Circulation 2004;110:3604–9. ´ ´ [22] Szczepanska M, Kozlik J, Skrzypczak J, et al. Oxidative stress may be a piece in the endometriosis puzzle. Fertil Steril 2003;79:1288–93. [23] Shanti A, Santanam N, Morales AJ, et al. Autoantibodies to markers of oxidative stress are elevated in women with endometriosis. Fertil Steril 1999;71:1115–8. [24] Tanner FC, Noll G, Boulanger CM, et al. Oxidized low density lipoproteins inhibit relaxations of porcine coronary arteries. Role of scavenger receptor and endothelium-derived nitric oxide. Circulation 1991;83:2012–20. [25] Chin JH, Azhar S, Hoffman BB. Inactivation of endothelial derived relaxing factor by oxidized lipoproteins. J Clin Invest 1992;89:10–8. [26] Ho WJ, Chen ST, Tsay PK, et al. Enhancement of endothelium-dependent flowmediated vasodilation in hyperthyroidism. Clin Endocrinol 2007;67:505–11. [27] Gazdag A, Nagy EV, Burman KD, et al. Improved endothelial function and lipid profile compensate for impaired hemostatic and inflammatory status in iatro-
genic chronic subclinical hyperthyroidism of thyroid cancer patients on L-t4 therapy. Exp Clin Endocrinol Diabetes 2010;118:381–7. [28] Danesh J, Collins R, Peto R. Chronic infections and coronary heart disease: is there a link? Lancet 1997;350:430–6. [29] Mattila KJ, Valtonen VV, Nieminen MS, et al. Role of infection as a risk factor for atherosclerosis, myocardial infarction, and stroke. Clin Infect Dis 1998;26:719–34. [30] Pretta S, Remorgida V, Abbamonte LH, et al. Atherosclerosis in women with endometriosis. Eur J Obstet Gynecol Reprod Biol 2007;132:226–31.