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Medicina Interna I (A.D., B.A., E.P., M.V., L.S., M.T., A.A.), Dipartimento di Scienze Cliniche e Biologiche, Università di Torino, Turin, Italy; Laboratorio Analisi (E.A.), Azienda Sanitaria Ospedaliera San Luigi, I-10043 Orbassano-Turin, Italy; and Endocrinologia (A.P.), Azienda Sanitaria Ospedaliera Santa Croce e Carle, I-12100 Cuneo, Italy
Address all correspondence and requests for reprints to: Andrea Dovio, Medicina Interna I, Dipartimento di Scienze Cliniche e Biologiche, Università degli Studi di Torino, Reg. Gonzole 10, 10043 Orbassano-Torino, Italy. E-mail: andrea.dovio{at}unito.it.
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Abstract |
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Objective: The aim of the study was to assess serum OPG and soluble receptor activator of nuclear factor-
B ligand (sRANKL) levels in CS and their possible relationship with coronary risk profile.
Design and Setting: We conducted a cross-sectional study at a tertiary referral center.
Patients: We studied 48 adult patients with CS and 48 age- and sex-matched controls. Twenty-six patients had pituitary-dependent CS; five patients had CS caused by ectopic ACTH secretion; and 17 patients had adrenal-dependent CS, accounted for by cortisol-secreting adenoma (n = 9), ACTH-independent macronodular bilateral adrenal hyperplasia (n = 4), or World Health Organization stage II cortisol-secreting carcinoma (n = 4). Patients underwent assessment of the absolute coronary risk and measurement of bone mineral density by dual-energy x-ray absorptiometry. Serum OPG and total sRANKL were measured by ELISA.
Results: Serum OPG (but not sRANKL) levels were significantly higher in CS patients than in controls (P < 0.01). In patients, serum OPG showed a positive correlation with age (r = 0.36; P = 0.01). OPG levels were higher in patients with the metabolic syndrome [median, 1262 (range, 199-2306) pg/ml vs. 867 (412–2479) pg/ml; P = 0.03], and showed a positive correlation with the absolute coronary risk (r = 0.36; P = 0.01). Serum OPG levels were higher in patients with pituitary-dependent CS in comparison with adrenal-dependent CS.
Conclusions: In patients with CS, serum OPG levels are increased and appear to be associated with coronary risk.
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Introduction |
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B ligand (RANKL), its receptor RANK, and its decoy receptor osteoprotegerin (OPG) has been followed by the acknowledgment of its major role in skeletal, vascular, and immune systems. In the bone microenvironment, the binding of RANKL to its cognate receptor RANK on cells of the hematopoietic lineage promotes differentiation and fusion of mononuclear osteoclast precursors to multinucleated cells and then activation of the multinucleated osteoclasts to resorb bone. OPG binds to RANKL, thus preventing RANKL-RANK interaction. The RANKL/OPG balance is the main determinant of osteoclast number and activity, and modulation of RANKL and OPG expression represents the common final pathway for a number of osteotropic signals involved in the regulation of bone resorption (1, 2). The availability of specific antibodies has made it possible to measure the levels of OPG and, more recently, soluble RANKL (sRANKL) in a number of biological fluids (3, 4). Early studies have unexpectedly found that serum OPG levels were increased in many patients with excess bone resorption. This was attributed to inadequate compensatory response to increased osteoclast activity, increased bone turnover, and associated diseases that could alter release or fate of OPG in the blood stream. However, data on the correlations of serum OPG levels with bone density and resorption markers were inconsistent (3, 4). Indeed, circulating OPG and sRANKL conceivably derive from several sources and do not merely reflect concentrations in the bone microenvironment (1, 3, 4).
The finding of arterial calcifications in OPG knockout mice was the first clue in the investigation of the role of this molecule in the vascular microenvironment. Subsequent in vitro studies have shown that OPG is expressed by both endothelial and vascular smooth muscle cells (VSMC), protects endothelial cells from apoptosis induced by the TNF-related apoptosis-inducing ligand, and possibly prevents RANKL-induced osteogenic differentiation of VSMC, a key event of vascular calcification (5). Finally, recent studies in mice deficient in both OPG and apolipoprotein E [OPG(–/–). ApoE(–/–) double knockout mice] have shown that OPG inhibits advanced plaque progression by preventing an increase in lesion size and calcification (6). Because of the enormous surface area of endothelium throughout the body, it is reasonable that endothelial cells, even if they release remarkably lower amounts of the molecule compared with cells of the osteoblastic lineage, significantly contribute to circulating OPG levels. Accordingly, the increase of serum OPG in patients with increased cardiovascular risk was suggested to reflect an inadequate compensatory response of the endothelium to damaging events (1, 4).
Patients with Cushing’s syndrome (CS) have a mortality rate four times higher than age- and sex-matched subjects, mainly due to cardiovascular events. Chronic hypercortisolism is associated with a cluster of cardiovascular risk factors such as arterial hypertension, impaired glucose tolerance or diabetes, central obesity, dyslipidemia, and hypercoagulability. Moreover, the prevalence of osteoporosis in adult patients with CS is approximately 50%, with 30–50% of patients experiencing pathological fractures (7, 8).
In vitro, glucocorticoids (GCs) inhibit OPG and stimulate RANKL expression in different cell types, including osteoblasts (1, 9). In vivo, short-term GC administration reduces serum OPG concentrations (10), whereas long-term administration of GCs was found to be associated with increased levels (11, 12). To date, however, only a single study has addressed the issue of serum OPG levels in CS (13), whereas to the best of our knowledge there are no data on serum sRANKL.
The aim of the present study was to measure serum OPG and sRANKL levels in a group of 48 adult patients with CS and to assess their possible relationships with coronary risk profile.
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Subjects and Methods |
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Forty-eight patients [19 men and 29 women; median age, 53 yr (range, 19–79 yr)] with overt CS referred to our center from 1995 to 2006 were studied. Exclusion criteria were history of diseases affecting bone, prolonged immobilization (>3 wk), and/or treatment with any drug documented to influence bone metabolism in humans in the previous 6 months.
Twenty-six patients had pituitary-dependent CS (or Cushing’s disease); five patients had CS caused by ectopic ACTH secretion; 17 patients had adrenal-dependent CS, accounted for by cortisol-secreting adenoma (n = 9), ACTH-independent macronodular bilateral adrenal hyperplasia (n = 4), or World Health Organization stage II cortisol-secreting carcinoma (n = 4). The ectopic ACTH secretion was caused by well-differentiated neuroendocrine tumors. All patients were evaluated in an active phase of the disease, and none of them was receiving drugs known to alter the hypothalamic-pituitary-adrenal axis. Twenty-eight patients were taking antihypertensive drugs as single agents or combination regimens: angiotensin I-converting enzyme inhibitors (n = 12), ß-blockers (n = 12), diuretics (n = 12), calcium channel blockers (n = 11), 
-1 adrenergic blockers (n = 4), and angiotensin II receptor blockers (n = 2). Seven patients were on statins, and two were on estrogens.
The diagnosis of CS was made on the basis of clinical features and standard hormonal and radiological criteria. For Cushing’s disease, the diagnosis was confirmed by finding a pituitary adenoma with positive ACTH staining at pathological examination or by the occurrence of postoperative adrenal insufficiency that lasted for at least 6 months. In all cases of ectopic ACTH syndrome and adrenal-dependent CS, the diagnosis was histologically confirmed by pathological findings and occurrence of postoperative adrenal insufficiency.
A total of 48 age- and sex-matched healthy subjects [19 men and 29 women; median age, 52.5 yr (range, 21–79 yr)] were recruited as controls among blood donors and healthy volunteers from 2002 to 2006. None of the controls were receiving medications or had medical conditions known to affect bone metabolism. Twelve control subjects were on antihypertensive drugs: diuretics (n = 4), angiotensin I-converting enzyme inhibitors (n = 2), angiotensin II receptor blockers (n = 3), ß-blockers (n = 2), and -1 adrenergic blockers (n = 1); three subjects were on statins.
Study protocol was prepared according to the Declaration of Helsinki and subsequent relevant integrations; all patients and controls gave written informed consent to participation, and approval by the local ethical committee was obtained.
Methods
Patients underwent complete physical examination, routine laboratory evaluation, dual-energy x-ray absorptiometry (DXA) on lumbar spine and femur, and endocrine work-up aimed to assess the function of the hypothalamic-pituitary-adrenal axis, as previously reported (14). Briefly, the patients underwent measurement of 1) serum cortisol at 0800 and 2400 h; 2) 24-h excretion of urinary free cortisol; 3) serum cortisol after overnight 1 and 8 mg dexamethasone; and 4) serum cortisol and plasma ACTH after ovine CRH test. Premenopausal women were studied in the early follicular phase of the menstrual cycle.
Assays
Blood samples for OPG and sRANKL measurement were drawn in the morning (0800–1000 h) from an antecubital vein. Serum OPG concentrations were measured by an ELISA developed in our laboratory using commercially available reagents (OPG DuoSet; R&D Systems, Abingdon, UK), with range 62.5–4000 pg/ml, minimum detectable concentration less than 30 pg/ml, and intra- and interassay coefficients of variation (CV) less than 5 and 13.5%, respectively. Serum total sRANKL concentrations were measured by a commercially available ELISA kit (Immundiagnostik, Bensheim, Germany) with range 133-3600 pg/ml, minimum detectable concentration less than 50 pg/ml, and intra- and interassay CV less than 5 and 10%, respectively.
Hormones were measured by RIA or immunoradiometric assay methods, using commercially available kits as previously described (14). All samples for an individual subject were determined in the same laboratory, in a single assay, in duplicate. Intra- and interassay CV for all hormones were less than 5 and 10%, respectively. Routine clinical chemistry variables were determined using standard methods.
DXA
Bone densitometry was performed by DXA using the Hologic QDR 4500 W instrument (Hologic, Inc., Waltham, MA; software version 9.03), with long-term CV of 0.46% at the spine using the Hologic Anthropometric Spine Phantom; and with short-term in vivo CV of 1 and 1.5% for spine and hip, respectively. Data were analyzed using absolute bone mineral density (BMD) values (grams per square centimeter), T-score and Z-score (referred to the manufacturer’s normative data for lumbar spine and to the National Health and Nutrition Examination Survey III dataset for the hip). The classical World Health Organization (WHO) criteria were used to define the conditions of normality, osteopenia, and osteoporosis (T-score above –1, between –1 and –2.5, or below –2.5, respectively).
Metabolic syndrome and cardiovascular risk
Metabolic syndrome was defined using National Cholesterol Education Program-Adult Treatment Panel III criteria (at least three of the following: fasting glucose 
110 mg/dl or treatment with antidiabetic drugs; blood pressure 130/85 mm Hg or treatment with antihypertensive agents; triglycerides 150 mg/dl; HDL < 40 mg/dl in women or < 50 mg/dl in men; and abdominal obesity, with waist circumference > 102 cm in men, > 88 cm in women) (15). Absolute coronary risk was estimated using a software developed by the Italian Society for the Study of Atherosclerosis, which estimates absolute coronary risk at 10 yr based on age, sex, blood pressure, total and HDL cholesterol, smoking status, diabetes, and left ventricular hypertrophy (16). Any subject with systolic blood pressure greater than 140 mm Hg, diastolic blood pressure greater than 90 mm Hg, or on antihypertensive treatment was categorized as hypertensive (17). Blood pressure was the average of two seated measurements taken with 5 min of rest by a physician using standardized techniques. Diabetes mellitus was diagnosed if a subject was on insulin or hypoglycemic agents or when the subject’s plasma glucose was greater than 126 mg/dl at fasting in at least two samples collected on different days (18). The values of absolute coronary risk were repeatedly found to correlate at the highest degree of significance (P < 0.0001) with those obtained with the use of the official risk chart developed by the Italian National Institute of Health, which estimates absolute vascular (not only coronary) risk at 10 yr. Because this latter chart does not apply to people under 35 yr and over 69 yr of age, the estimates of absolute coronary risk were used for statistical analysis.
Statistical analysis
Database management and all statistical analyses were performed by Statistica 6.0 (Statsoft Inc., Tulsa, OK). To assess differences in discrete variables, 
2 test was used. Normality of continuous data was assessed by the Wilk-Shapiro’s test. Because most continuous variables showed nonnormal distribution, differences and correlations were analyzed by two-tailed Mann-Whitney U test and Spearman r coefficient, respectively. Level of statistical significance was set at P < 0.05.
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Results |
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. Serum OPG levels were significantly higher in CS patients than in controls, whereas significance was not attained for differences in sRANKL (Fig. 1) and sRANKL/OPG ratio. Because OPG values are markedly affected by preanalytical factors, including duration of storage (3, 4), the same analysis was also performed in a subset of 29 patients whose blood samples had been collected between 2001 and 2006. A significant increase in CS patients was confirmed [median 1052 (range 199-2479) pg/ml vs. 678 (349–1441) pg/ml; P = 0.01].
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The 19 patients with the metabolic syndrome had higher serum OPG levels than the remainders [1262 (199–2306) pg/ml vs. 867 (412–2479) pg/ml; P = 0.03], with no difference in indices of cortisol secretion. Serum OPG but not sRANKL levels showed a significant positive correlation with the absolute coronary risk (r = 0.36, P = 0.01; Fig. 2).
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Serum OPG levels were higher in patients with pituitary-dependent syndrome in comparison with those with adrenal-dependent syndrome, whereas no difference in sRANKL was observed (Fig. 3). Notably, the two groups were comparable for age, cortisol levels, and coronary risk, but differed for lumbar spine densitometric parameters (Table 2).
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Discussion |
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These observations are consistent with the recent opinion that circulating OPG is a novel marker for the development/progression of atherosclerotic disease (1, 3, 4, 5, 19, 20). OPG levels have been found to be increased in patients with coronary artery disease, acute myocardial infarction, increased carotid intima-media thickness, and coronary and aortic calcifications (4). As a general conclusion, circulating OPG levels have been considered to predict cardiovascular mortality in elderly women and cardiovascular disease in the general population (19, 20). Not surprisingly, they have been found to be associated with a number of established cardiovascular risk factors (including age, diabetes, hypertension, smoking, markers of systemic inflammation, chronic infection, and homocysteine) (4).
There is also increasing evidence that GCs at supraphysiological concentrations have a deleterious action on vascular health either by directly promoting the development of atheroma or by inducing intermediate risk factors. Patients with overt CS exhibit increased vascular morbidity and mortality, with acute myocardial infarction being the most common cause of death in multiple case series (21). Pertinently, in a large cross-sectional study of healthy participants, levels of salivary cortisol were significantly associated with intima-media thickness in the carotid artery (22). Notwithstanding the complexity of the process of atherogenesis, it is nowadays held that sustained GC excess, clinically apparent or inapparent, is proatherogenic (23). In the most recent years, cellular and molecular mechanisms supporting the concept of GCs as direct promoters of atherogenesis are being disclosed (24, 25). Interestingly for the analogy with OPG, bimodal regulation by GCs as a function of concentrations and duration of exposure has been suggested to be crucial for another, allegedly important factor in the control of vascular immune reactions and bone remodeling process as well, the macrophage migration inhibitory factor (26, 27).
In the present study, serum OPG levels positively correlated with morning serum cortisol, similarly to the findings by Ueland et al. (13). In vitro, GCs inhibit OPG expression in a variety of cell lines (1, 2, 9). In patients given GCs, although short-term administration reduces serum OPG (10), prolonged administration is more frequently associated with increased levels (11, 12). The results of our study suggest an explanation for this biphasic pattern: early decrease due to direct effects on OPG synthesis and release by the physiological major source, i.e. cells of the osteoblastic lineage, and subsequent increase due to long-term vascular damage overriding the initial effects.
GCs also inhibit GH secretion (28). Notably, increased OPG protein and gene expression has been demonstrated in cortical bone explants after GH replacement therapy (29); in contrast, GH did not affect OPG expression in human VSMC (30), and no changes in serum OPG were seen during GH replacement therapy in most studies (29). Therefore, inhibited GH secretion, although playing a role in the pathogenesis of GC-induced osteoporosis, is unlikely to account for increased serum OPG levels in CS patients.
We did not find significant correlations of OPG, sRANKL, and their ratio with BMD parameters. A number of explanations are plausible. First, BMD values in patients with secondary osteoporosis are the result of a number of factors, which can confound the effects of the underlying disease. Second, GCs are known to affect bone quality and not only quantity, which is assessed by DXA measurement (8). Third, previous data on relationships between circulating OPG and BMD were inconsistent (4, 31, 32, 33, 34).
We and others have reported that bone loss was more severe in patients bearing a cortisol-secreting adenoma vs. those who had a pituitary-dependent hypercortisolism (35, 36). In the present work, we found that serum OPG levels were higher in pituitary-dependent than in adrenal-dependent CS patients. Whatever might be the source of circulating OPG in these cases, its putative role in the pathogenesis of the bone loss occurring in CS as a function of different etiologies awaits further investigation.
A number of limitations of the present study should be noted. First, data on serum OPG and sRANKL after surgery were available only for a small subgroup (data not shown), hence the effect of recovery of normal adrenal function remains to be elucidated. Second, we did not assess more direct indexes of vascular disease (such as carotid intima-media thickness, or carotid/iliofemoral atherosclerotic plaque occurrence). Third, we did not measure bone turnover markers, which presumably better reflect the current formation and resorption activities; therefore, a relationship between serum OPG and bone metabolism in these patients may not be definitively excluded.
In conclusion, we have shown that serum OPG levels are increased in CS patients with respect to age- and sex-matched controls. The apparent increment is likely to be related to the metabolic and cardiovascular effects of sustained hypercortisolism and may be a marker for the higher coronary risk of patients with CS.
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Acknowledgments |
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Footnotes |
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Disclosure Statement: B.A., E.P., A.P., E.A., and M.T. have nothing to declare. A.D. and M.V. received lecture fees from Procter & Gamble and Merck Sharp & Dohme. L.S. received consulting fees from Adaltis. A.A. received lecture fees from Novartis and Procter & Gamble.
First Published Online February 27, 2007
Abbreviations: BMD, Bone mineral density; CS, Cushing’s syndrome; CV, coefficient(s) of variation; DXA, dual-energy x-ray absorptiometry; GC, glucocorticoid(s); OPG, osteoprotegerin; RANK, receptor activator of nuclear factor-B; RANKL, RANK ligand; sRANKL, soluble RANKL; VSMC, vascular smooth muscle cells.
Received October 19, 2006.
Accepted February 16, 2007.
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References |
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B ligand: clinical utility in metabolic bone disease assessment. J Clin Endocrinol Metab 90:6323–6331. Diabetologia 48:561–568[CrossRef][Medline]
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