An Increased Osteoprotegerin Serum Release Characterizes the Early Onset of Diabetes Mellitus and May Contribute to Endothelial Cell Dysfunction
2006; Elsevier BV; Volume: 169; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2006.060398
ISSN1525-2191
AutoresPaola Secchiero, Federica Corallini, Assunta Pandolfi, Agostino Consoli, Riccardo Candido, Bruno Fabris, Claudio Celeghini, Silvano Capitani, Giorgio Zauli,
Tópico(s)Bone health and treatments
ResumoSerum osteoprotegerin (OPG) is significantly increased in diabetic patients, prompting expanded investigation of the correlation between OPG production/release and glycemic levels. Serum levels of OPG, but not of its cognate ligand receptor activator of nuclear factor-κB ligand (RANKL), were significantly increased in type 2 diabetes mellitus patients compared with healthy blood donors. Serum OPG was also significantly elevated in a subgroup of recently diagnosed diabetic patients (within 2 years). The relationship between serum OPG and diabetes mellitus onset was next investigated in apoE-null and littermate mice. Serum OPG increased early after diabetes induction in both mouse strains and showed a positive correlation with blood glucose levels and an inverse correlation with the levels of free (OPG-unbound) RANKL. The in vitro addition of tumor necrosis factor-α to human vascular endothelial cells, but not human peripheral blood mononuclear cells, markedly enhanced OPG release in culture. In contrast, high glucose concentrations did not modulate OPG release when used alone or in association with tumor necrosis factor-α. Moreover, the ability of soluble RANKL to activate the extracellular signal-regulated kinase/mitogen-activated protein kinase and endothelial nitric-oxide synthase pathways in endothelial cells was neutralized by preincubation with recombinant OPG. Altogether, these findings suggest that increased OPG production represents an early event in the natural history of diabetes mellitus, possibly contributing to disease-associated endothelial cell dysfunction. Serum osteoprotegerin (OPG) is significantly increased in diabetic patients, prompting expanded investigation of the correlation between OPG production/release and glycemic levels. Serum levels of OPG, but not of its cognate ligand receptor activator of nuclear factor-κB ligand (RANKL), were significantly increased in type 2 diabetes mellitus patients compared with healthy blood donors. Serum OPG was also significantly elevated in a subgroup of recently diagnosed diabetic patients (within 2 years). The relationship between serum OPG and diabetes mellitus onset was next investigated in apoE-null and littermate mice. Serum OPG increased early after diabetes induction in both mouse strains and showed a positive correlation with blood glucose levels and an inverse correlation with the levels of free (OPG-unbound) RANKL. The in vitro addition of tumor necrosis factor-α to human vascular endothelial cells, but not human peripheral blood mononuclear cells, markedly enhanced OPG release in culture. In contrast, high glucose concentrations did not modulate OPG release when used alone or in association with tumor necrosis factor-α. Moreover, the ability of soluble RANKL to activate the extracellular signal-regulated kinase/mitogen-activated protein kinase and endothelial nitric-oxide synthase pathways in endothelial cells was neutralized by preincubation with recombinant OPG. Altogether, these findings suggest that increased OPG production represents an early event in the natural history of diabetes mellitus, possibly contributing to disease-associated endothelial cell dysfunction. Receptor activator of nuclear factor (NF)-κB ligand (RANKL) is a member of the tumor necrosis factor (TNF) family of cytokines, which exists either as type II membrane or as soluble protein.1Anderson DM Maraskovsky E Billingsley WL Dougall WC Tometsko ME Roux ER Teepe MC DuBose RF Cosman D Galibert L A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function.Nature. 1997; 390: 175-179Crossref PubMed Scopus (1960) Google Scholar RANKL was originally described as being expressed by activated T lymphocytes and osteoblasts, and it has been involved in the interaction between T lymphocytes and dendritic cells, osteoclast differentiation from monocytic precursor cells, and activation of mature osteoclasts.1Anderson DM Maraskovsky E Billingsley WL Dougall WC Tometsko ME Roux ER Teepe MC DuBose RF Cosman D Galibert L A homologue of the TNF receptor and its ligand enhance T-cell growth and dendritic-cell function.Nature. 1997; 390: 175-179Crossref PubMed Scopus (1960) Google Scholar, 2Lacey DL Timms E Tan HL Kelley MJ Dunstan CR Burgess T Elliott R Colombero A Elliott G Scully S Hsu H Sullivan J Hawkins N Davy E Capparelli C Eli A Qian YX Kaufman S Sarosi I Shalhoub V Senaldi G Guo J Delaney J Boyle WJ Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4656) Google Scholar, 3Hsu H Lacey DL Dunstan CR Solovyev I Colombero A Timms E Tan HL Elliott G Kelley MJ Sarosi I Wang L Xia XZ Elliott R Chiu L Black T Scully S Capparelli C Morony S Shimamoto G Bass MB Boyle WJ Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.Proc Natl Acad Sci USA. 1999; 96: 3540-3545Crossref PubMed Scopus (1428) Google Scholar, 4Dougall WC Glaccum M Charrier K Rohrbach K Brasel K De Smedt T Daro E Smith J Tometsko ME Maliszewski CR Armstrong A Shen V Bain S Cosman D Anderson D Morrissey PJ Peschon JJ Schuh J RANK is essential for osteoclast and lymph node development.Genes Dev. 1999; 13: 2412-2418Crossref PubMed Scopus (1210) Google Scholar, 5Kong YY Yoshida H Sarosi I Tan HL Timms E Capparelli C Morony S Oliveira-dos-Santos AJ Van G Itie A Khoo W Wakeham A Dunstan CR Lacey DL Mak TW Boyle WJ Penninger JM OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.Nature. 1999; 397: 315-323Crossref PubMed Scopus (2887) Google Scholar, 6Wong BR Josien R Choi Y TRANCE is a TNF family member that regulates dendritic cell and osteoclast function.J Leukoc Biol. 1999; 65: 715-724Crossref PubMed Scopus (199) Google Scholar Two receptors for RANKL have been identified: transmembrane RANK and soluble osteoprotegerin (OPG).2Lacey DL Timms E Tan HL Kelley MJ Dunstan CR Burgess T Elliott R Colombero A Elliott G Scully S Hsu H Sullivan J Hawkins N Davy E Capparelli C Eli A Qian YX Kaufman S Sarosi I Shalhoub V Senaldi G Guo J Delaney J Boyle WJ Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiation and activation.Cell. 1998; 93: 165-176Abstract Full Text Full Text PDF PubMed Scopus (4656) Google Scholar, 3Hsu H Lacey DL Dunstan CR Solovyev I Colombero A Timms E Tan HL Elliott G Kelley MJ Sarosi I Wang L Xia XZ Elliott R Chiu L Black T Scully S Capparelli C Morony S Shimamoto G Bass MB Boyle WJ Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.Proc Natl Acad Sci USA. 1999; 96: 3540-3545Crossref PubMed Scopus (1428) Google Scholar, 5Kong YY Yoshida H Sarosi I Tan HL Timms E Capparelli C Morony S Oliveira-dos-Santos AJ Van G Itie A Khoo W Wakeham A Dunstan CR Lacey DL Mak TW Boyle WJ Penninger JM OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.Nature. 1999; 397: 315-323Crossref PubMed Scopus (2887) Google Scholar RANK mRNA is ubiquitously expressed in human tissues, but RANK protein expression has been characterized only in normal dendritic cells, CD4 and CD8 T lymphocytes, osteoclast monocytic precursors, and endothelial cells, suggesting that expression of this protein is posttranscriptionally regulated.6Wong BR Josien R Choi Y TRANCE is a TNF family member that regulates dendritic cell and osteoclast function.J Leukoc Biol. 1999; 65: 715-724Crossref PubMed Scopus (199) Google Scholar For the purpose of this study, it is noteworthy that, by interacting with RANK, RANKL induces a variety of biological effects on endothelial cells, such as promotion of cell survival and angiogenesis.7Kim YM Kim YM Lee YM Kim HS Kim JD Choi Y Kim KW Lee SY Kwon YG TNF-related activation-induced cytokine (TRANCE) induces angiogenesis through the activation of Src and phospholipase C (PLC) in human endothelial cells.J Biol Chem. 2002; 277: 6799-6805Crossref PubMed Scopus (107) Google Scholar, 8Kim HE Shin HS Kwak HJ Ahn KY Kim JH Lee HJ Lee MS Lee ZH Koh GY RANKL regulates endothelial cell survival through the phosphatidylinositol 3′-kinase/Akt signal transduction pathway.FASEB J. 2003; 17: 2163-2165PubMed Google Scholar, 9Min JK Kim YM Kim YM Kim EC Gho YS Kang IJ Lee SY Kong YY Kwon YG Vascular endothelial growth factor up-regulates expression of receptor activator of NF-kappa B (RANK) in endothelial cells. Concomitant increase of angiogenic responses to RANK ligand.J Biol Chem. 2003; 278: 39548-39557Crossref PubMed Scopus (101) Google Scholar Although the affinity of RANKL for OPG is weaker than that for RANK,5Kong YY Yoshida H Sarosi I Tan HL Timms E Capparelli C Morony S Oliveira-dos-Santos AJ Van G Itie A Khoo W Wakeham A Dunstan CR Lacey DL Mak TW Boyle WJ Penninger JM OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.Nature. 1999; 397: 315-323Crossref PubMed Scopus (2887) Google Scholar when present at high concentrations soluble OPG prevents RANKL interaction with transmembrane RANK, thus acting as a decoy receptor.3Hsu H Lacey DL Dunstan CR Solovyev I Colombero A Timms E Tan HL Elliott G Kelley MJ Sarosi I Wang L Xia XZ Elliott R Chiu L Black T Scully S Capparelli C Morony S Shimamoto G Bass MB Boyle WJ Tumor necrosis factor receptor family member RANK mediates osteoclast differentiation and activation induced by osteoprotegerin ligand.Proc Natl Acad Sci USA. 1999; 96: 3540-3545Crossref PubMed Scopus (1428) Google Scholar, 5Kong YY Yoshida H Sarosi I Tan HL Timms E Capparelli C Morony S Oliveira-dos-Santos AJ Van G Itie A Khoo W Wakeham A Dunstan CR Lacey DL Mak TW Boyle WJ Penninger JM OPGL is a key regulator of osteoclastogenesis, lymphocyte development and lymph-node organogenesis.Nature. 1999; 397: 315-323Crossref PubMed Scopus (2887) Google Scholar It has been shown that OPG is produced by a wide range of tissues, including the cardiovascular system, and that OPG levels are particularly high in aortic and renal arteries.10Collin-Osdoby P Regulation of vascular calcification by osteoclast regulatory factors RANKL and osteoprotegerin.Circ Res. 2004; 95: 1046-1057Crossref PubMed Scopus (408) Google Scholar, 11Schoppet M Al-Fakhri N Franke FE Katz N Barth PJ Maisch B Preissner KT Hofbauer LC Localization of osteoprotegerin, tumor necrosis factor-related apoptosis-inducing ligand, and receptor activator of nuclear factor-kB ligand in Monckeberg's sclerosis and atherosclerosis.J Clin Endocrinol Metab. 2004; 89: 4104-4112Crossref PubMed Scopus (179) Google Scholar, 12Olesen P Ledet T Rasmussen LM Arterial osteoprotegerin: increased amounts in diabetes and modifiable synthesis from vascular smooth muscle cells by insulin and TNF-alpha.Diabetologia. 2005; 48: 561-568Crossref PubMed Scopus (119) Google Scholar Interestingly, different groups of investigators have reported that serum OPG is significantly increased in both type 1 and type 2 diabetic patients,13Galluzzi F Stagi S Salti R Toni S Piscitelli E Simonini G Falcini F Chiarelli F Osteoprotegerin serum levels in children with type 1 diabetes: a potential modulating role in bone status.Eur J Endocrinol. 2005; 153: 879-885Crossref PubMed Scopus (66) Google Scholar, 14Rasmussen LM Tarnow L Hansen TK Parving HH Flyvbjerg A Plasma osteoprotegerin levels are associated with glycaemic status, systolic blood pressure, kidney function and cardiovascular morbidity in type 1 diabetic patients.Eur J Endocrinol. 2006; 154: 75-81Crossref PubMed Scopus (138) Google Scholar, 15Browner WS Lui LY Cummings SR Associations of serum osteoprotegerin levels with diabetes, stroke, bone density, fractures, and mortality in elderly women.J Clin Endocrinol Metab. 2001; 86: 631-637Crossref PubMed Scopus (482) Google Scholar, 16Knudsen ST Foss CH Poulsen PL Andersen NH Mogensen CE Rasmussen LM Increased plasma concentrations of osteoprotegerin in type 2 diabetic patients with microvascular complications.Eur J Endocrinol. 2003; 149: 39-42Crossref PubMed Scopus (153) Google Scholar, 17Avignon A Sultan A Piot C Elaerts S Cristol JP Dupuy AM Osteoprotegerin is associated with silent coronary artery disease in high-risk but asymptomatic type 2 diabetic patients.Diabetes Care. 2005; 28: 2176-2180Crossref PubMed Scopus (95) Google Scholar, 18Anand DV Lahiri A Lim E Hopkins D Corder R The relationship between plasma osteoprotegerin levels and coronary artery calcification in uncomplicated type 2 diabetic subjects.J Am Coll Cardiol. 2006; 47: 1850-1857Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar as well as in both diabetic and nondiabetic patients affected by coronary artery disease.17Avignon A Sultan A Piot C Elaerts S Cristol JP Dupuy AM Osteoprotegerin is associated with silent coronary artery disease in high-risk but asymptomatic type 2 diabetic patients.Diabetes Care. 2005; 28: 2176-2180Crossref PubMed Scopus (95) Google Scholar, 18Anand DV Lahiri A Lim E Hopkins D Corder R The relationship between plasma osteoprotegerin levels and coronary artery calcification in uncomplicated type 2 diabetic subjects.J Am Coll Cardiol. 2006; 47: 1850-1857Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar, 19Jono S Ikari Y Shioi A Mori K Miki T Hara K Nishizawa Y Serum osteoprotegerin levels are associated with the presence and severity of coronary artery disease.Circulation. 2002; 106: 1192-1194Crossref PubMed Scopus (454) Google Scholar, 20Schoppet M Sattler AM Juergen R Schaefer JR Herzum M Maisch B Hofbauer LC Increased osteoprotegerin serum levels in men with coronary artery disease.J Clin Endocrinol Metab. 2003; 88: 1024-1028Crossref PubMed Scopus (300) Google Scholar Moreover, it has been demonstrated that up-regulated serum OPG levels have a negative prognostic value in heart failure after acute myocardial infarction as well as in patients affected by abdominal aortic aneurysm.21Ueland T Jemtland R Godang K Kjekshus J Hognestad Omland T Squire IB Gullestad L Bollerslev J Dickstein K Aukrust P Prognostic value of osteoprotegerin in heart failure after acute myocardial infarction.J Am Coll Cardiol. 2004; 44: 1970-1976Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar, 22Barreto DV Barreto FC Carvalho AB Cuppari L Cendoroglo M Draibe SA Moyses RM Neves KR Jorgetti V Blair A Guiberteau R Fernandes Canziani ME Coronary calcification in hemodialysis patients: the contribution of traditional and uremia-related risk factors.Kidney Int. 2005; 67: 1576-1582Crossref PubMed Scopus (123) Google Scholar, 23Moran CS McCann M Karan M Norman P Ketheesan N Golledge J Association of osteoprotegerin with human abdominal aortic aneurysm progression.Circulation. 2005; 111: 3119-3125Crossref PubMed Scopus (139) Google Scholar Interestingly, it has also been shown that the levels of free RANKL are significantly decreased in the sera of patients affected by coronary artery disease24Schoppet M Schaefer JR Hofbauer LC Low serum levels of soluble RANK ligand are associated with the presence of coronary artery disease in men.Circulation. 2003; 107: e76Crossref PubMed Google Scholar as well as in the endomyocardium in transplant coronary artery disease.25Ueland T Gullestad L Simonsen S Endresen K Scott H Froland SS Geiran O Fiane AE Aukrust P Decreased endomyocardial RANKL expression in transplant coronary artery disease.Transplantation. 2006; 81: 1467-1470Crossref PubMed Scopus (3) Google Scholar The aim of this study was to investigate whether serum OPG elevation represents an early or a late event in the natural history of diabetes mellitus and to investigate the correlation between OPG production/release and glycemic levels both in vivo and in vitro. Serum samples were obtained from 88 patients with type 2 diabetes mellitus and 41 control patients who had no metabolic disease. The study was approved by the "G. D'Annunzio" University Ethical Committee, and the consent was obtained from patients after full explanation of the procedure and its purpose, in accordance with Declaration of Helsinki of 1975. Characteristics of the patients are summarized in Table 1. In particular, 20 of 88 diabetic patients had microvascular complications: 12 had background diabetic retinopathy, four had preproliferative diabetic retinopathy, and four had undergone argon laser treatment for proliferative diabetic retinopathy. Two of the patients with background retinopathy also exhibited diabetic nephropathy (proteinuria >300 mg/24 hours). Only four patients had clinically manifest diabetic macroangiopathy (two had experienced a myocardial infarction, one had undergone coronary angioplasty, and one had undergone coronary artery bypass graft).Table 1Characteristics of the Diabetic Study GroupNumber of cases88Gender (M/F)40/48Age*Median and range. (years)65 (58 to 75)BMI*Median and range.30 (22.4 to 41.5)HbA1c*Median and range. (%)7.6 (5.3 to 11.5)Pharmacological therapy†Metformin, Gliclazide, Rosiglitazone, Glibenclamide./diet60/28Patients with vascular complications24Duration diabetes*Median and range. (years from diagnosis)10 (<1 to 32)* Median and range.† Metformin, Gliclazide, Rosiglitazone, Glibenclamide. Open table in a new tab Animal care and treatments were conducted in conformity with institutional guidelines in compliance with national and international laws and policies (European Economic Community, Council Directive 86/609, OJL 358, December 12, 1987). Sixteen apoE-null (ApoEtm1Unc) mice, 6 weeks old, and eight littermates (C57Black/6J strain) were rendered diabetic by five daily intraperitoneal injections of streptozotocin (STZ; Sigma Chemical Co., St. Louis, MO) at a dose of 55 mg/kg. Control apoE-null mice (n = 10) and littermates (n = 8) received citrate buffer alone and were processed in parallel to the diabetic mice. The animals had unrestricted access to water and were maintained on a 12-hour light-dark cycle in a nonpathogen-free environment on standard mouse chow (Harlan Nossan Correzzana, Milan, Italy). Serum glucose, total cholesterol, high-density lipoprotein, and triglyceride concentrations were determined by an autoanalyzer technique (Hitachi 717; Tokyo, Japan). For the histological examination, after 3 months, the animals were anesthetized by an intraperitoneal injection of pentobarbital sodium (60 mg/kg body wt; Boehringer, Ingelheim, Germany). The distribution and extent of atherosclerotic lesions in apoE-null mice were evaluated by the en face analysis, after staining with Sudan IV-Herxheimer's solution (Sigma Chemical Co.), as previously described.26Candido R Allen TJ Lassila M Cao Z Thallas V Cooper ME Jandeleit-Dahm KA Irbesartan but not amlodipine suppresses diabetes-associated atherosclerosis.Circulation. 2004; 109: 1536-1542Crossref PubMed Scopus (191) Google Scholar Aortic segments were then embedded in paraffin, and 4-μm-thick cross-sectional serial sections were stained with hematoxylin and eosin to evaluate the atherosclerotic lesion complexity. Human OPG and RANKL levels were measured in serum samples as well as in cell culture supernatants using sandwich-type enzyme-linked immunosorbent assay (ELISA) kits according to the manufacturers' instructions. The human OPG ELISA kit was purchased from Alexis Biochemicals (Lausen, Switzerland), and human RANKL kits were purchased from Apotech (Epalinges, Switzerland) and Biomedica (Vienna, Austria). Mouse RANKL and OPG serum levels were measured in sera from apoE-null and C57Black littermate mice using ELISA kits purchased from R&D Systems (Minneapolis, MN). The results were read at an optical density of 450 nm using an Anthos 2010 ELISA reader (Anthos Labtec Instruments Ges.m.b.H, Wals/Salzburg, Austria). Measurements were done in duplicates. Of note, the ELISA assay for human RANKL from Apotech uses the two-site sandwich technique with two selected antibodies that bind to human sRANKL and OPG, allowing the determination of total (both free and OPG-bound) RANKL. On the other hand, the ELISA assay for human RANKL from Biomedica, as well as the assay for mouse RANKL (R&D Systems), detects only uncomplexed free RANKL. TNF-α, glucose, and insulin were purchased from Sigma; interleukin (IL)-1β was from Roche Diagnostics (Mannheim, Germany); recombinant OPG was from R&D Systems; recombinant RANKL was from Alexis. For Western blot analyses, the following antibodies (Abs) were used: anti-extracellular signal-regulated kinase (ERK) 1/2, anti-phospho-ERK1/2 (both from New England Biolabs, Beverly, MA), anti-phospho-endothelial nitric-oxide synthase (eNOS) (P-Ser1177; Cell Signaling Technology, Beverly, MA), anti-eNOS/NOS type III (BD Transduction Laboratories, Lexington, KY), and anti-tubulin (Sigma). Primary human umbilical vein endothelial cells (HUVECs), obtained from BioWhittaker (Walkersville, MD), were used between passages 3 and 6 in vitro. Cells were grown on gelatin-coated tissue culture plates in M199 endothelial growth medium (BioWhittaker) supplemented with 20% fetal bovine serum (Life Technologies, Inc., Gaithersburg, MD), 10 μg/ml heparin, and 50 μg/ml endothelial cell growth factor (Sigma), as previously described.27Secchiero P Corallini F di Iasio MG Gonelli A Barbarotto E Zauli G TRAIL counteracts the proadhesive activity of inflammatory cytokines in endothelial cells by down-modulating CCL8 and CXCL10 chemokine expression and release.Blood. 2005; 105: 3413-3419Crossref PubMed Scopus (95) Google Scholar Human peripheral blood mononuclear cells (PBMCs) from healthy normal donors were separated by gradient centrifugation with lymphocyte cell separation medium (Cedarlane Laboratories, Hornby, ON, Canada) and seeded at a density of 1 to 5 × 106 cells/well. For macrophage cultures, after incubation for 18 hours, nonadherent PBMCs were removed, and remaining adherent cells were maintained in RPMI medium containing 10% fetal bovine serum and 50 ng/ml human macrophage-colony-stimulating factor (PeproTech, London, UK). Expression of macrophagic markers was documented by flow cytometry using phycoerythrin-conjugated anti-CD14 (Immunotech, Marseille, France) and anti-CD36 antibodies (BD Pharmingen, San Diego, CA), and fluorescein isothiocyanate-conjugated anti-CD64 antibody (Immunotech). Cells were treated with glucose (30 mmol/L), insulin (1 μmol/L), or scalar concentration of inflammatory cytokines (TNF-α or IL-1β; 1 to 100 pg/ml) or of STZ (0.2 to 5 μmol/L). Supernatants were harvested at 24 and 72 hours after the treatments and analyzed for OPG and RANKL levels. For immunoblot experiments, HUVECs were plated in 10-cm dishes and grown at subconfluence before treatments. To minimize activation by serum, HUVECs were subject to partial fetal bovine serum reduction (0.5%) and growth factor withdrawal for 18 hours before the addition of RANKL, used alone or in combination with OPG. The optimal concentrations for RANKL (10 ng/ml) and OPG (20 ng/ml) were determined in preliminary experiments in which HUVECs were exposed to serial dilutions (0.1 to 100 ng/ml) of the molecules. For protein preparation, cells were harvested in lysis buffer containing 1% Triton X-100, Pefablock (1 mmol/L), aprotinin (10 μg/ml), pepstatin (1 μg/ml), leupeptin (10 μg/ml), NaF (10 mmol/L), and Na3VO4 (1 mmol/L). Protein determination was performed by Bradford assay (Bio-Rad, Richmond, CA). Equal amounts of protein (50 μg) for each sample were migrated in acrylamide gels and blotted onto nitrocellulose filters. Blotted filters were probed with antibodies for the phosphorylated ERK1/2 and eNOS. After incubation with peroxidase-conjugated anti-rabbit or anti-mouse IgG, specific reactions were revealed with the enhanced chemiluminescence reagent detection system (DuPont-NEN, Boston, MA). Membranes were stripped by incubation in Re-Blot 1X antibody stripping solution (Chemicon Int., Temecula, CA) and reprobed for the respective total ERK1/2 and eNOS protein content and for tubulin levels, for verifying loading evenness. Densitometric values were expressed in arbitrary units and estimated by the ImageQuant software (Molecular Dynamics, Piscataway, NJ). Multiple film exposures were used to verify the linearity of the samples analyzed and avoid saturation of the film. For NO-dependent guanosine 3′,5′-cyclic monophosphate (cGMP) measurement, HUVECs were seeded in standard 96-well plates, incubated overnight at standard conditions, and subsequently treated, as indicated, for 30 minutes at 37°C in culture medium containing 0.6 mmol/L 3-isobutyl-1-methylxanthine. After cell lysis, cGMP levels were measured using an enzyme-immunoassay kit (cGMP EIA system; Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK) according to the manufacturer's instructions. The median, minimum, and maximum values were calculated for each group of data obtained from both human and mouse serum samples. Box plots were used to show the median, minimum, and maximum values and 25th to 75th percentiles. The results were evaluated by using Student's t-test and the Mann-Whitney rank-sum test. Correlation coefficients were calculated by the Spearman's method. Statistical significance was defined as P < 0.05. In the first group of experiments, the serum levels of OPG and RANKL were examined in 88 type 2 diabetic patients in comparison to 41 healthy blood donors. The serum levels of OPG were significantly (P < 0.05) increased in diabetic patients (mean ± SD, 130 ± 41 pg/ml) with respect to sex- and aged-matched normal blood donors (mean ± SD, 80 ± 29 pg/ml) (Figure 1A). On the other hand, the serum concentration of total (free plus OPG-bound) RANKL did not show any significant variation between diabetic patients (mean ± SD, 79 ± 116 pg/ml) and normal controls (mean ± SD, 69 ± 75) (Figure 1B). Of note, in a limited group of diabetic patients (n = 40) and healthy controls (n = 22), we also examined the levels of free RANKL using an ELISA kit, which specifically recognizes free RANKL (ie, unbound to OPG). As shown in Figure 1C, the levels of free RANKL were significantly (P < 0.05) decreased in diabetic patients (mean ± SD, 5.2 ± 4 pg/ml) with respect to normal blood donors (mean ± SD, 10.5 ± 6 pg/ml), in line with the concomitant increase of serum OPG. A significant correlation (r = 0.29, P < 0.05) was observed between serum OPG levels and the history of diabetes mellitus. In particular, because diabetic patients were clustered into two subgroups, ie, newly diagnosed diabetic patients (within 2 years from diagnosis) and patients with a longer history of diabetes (≥6 years from diagnosis), it is remarkable that recently diagnosed diabetic patients also showed significantly (P < 0.05) greater OPG serum levels with respect to normal controls (Figure 2A). Patients with a longer history of diabetes mellitus showed higher levels of OPG with respect to either recently diagnosed patients (P < 0.05) and normal controls (Figure 2A). Of note, patients with a long history of diabetes mellitus comprised most of the patients with documented microvascular and macrovascular complications (Table 1). The possibility that the increased serum OPG levels detected in patients with a long history of diabetes mellitus merely reflected an advanced age of these patients was excluded, because no significant correlation was observed between serum OPG and age of the patients (r = 0.05, P > 0.05) (Figure 2B). Elevated levels of serum OPG have been reported in patients affected by either type 1 or type 2 diabetes mellitus.13Galluzzi F Stagi S Salti R Toni S Piscitelli E Simonini G Falcini F Chiarelli F Osteoprotegerin serum levels in children with type 1 diabetes: a potential modulating role in bone status.Eur J Endocrinol. 2005; 153: 879-885Crossref PubMed Scopus (66) Google Scholar, 14Rasmussen LM Tarnow L Hansen TK Parving HH Flyvbjerg A Plasma osteoprotegerin levels are associated with glycaemic status, systolic blood pressure, kidney function and cardiovascular morbidity in type 1 diabetic patients.Eur J Endocrinol. 2006; 154: 75-81Crossref PubMed Scopus (138) Google Scholar, 15Browner WS Lui LY Cummings SR Associations of serum osteoprotegerin levels with diabetes, stroke, bone density, fractures, and mortality in elderly women.J Clin Endocrinol Metab. 2001; 86: 631-637Crossref PubMed Scopus (482) Google Scholar, 16Knudsen ST Foss CH Poulsen PL Andersen NH Mogensen CE Rasmussen LM Increased plasma concentrations of osteoprotegerin in type 2 diabetic patients with microvascular complications.Eur J Endocrinol. 2003; 149: 39-42Crossref PubMed Scopus (153) Google Scholar, 17Avignon A Sultan A Piot C Elaerts S Cristol JP Dupuy AM Osteoprotegerin is associated with silent coronary artery disease in high-risk but asymptomatic type 2 diabetic patients.Diabetes Care. 2005; 28: 2176-2180Crossref PubMed Scopus (95) Google Scholar, 18Anand DV Lahiri A Lim E Hopkins D Corder R The relationship between plasma osteoprotegerin levels and coronary artery calcification in uncomplicated type 2 diabetic subjects.J Am Coll Cardiol. 2006; 47: 1850-1857Abstract Full Text Full Text PDF PubMed Scopus (178) Google Scholar Therefore, to further analyze whether the serum levels of OPG were affected by hyperglycemia and/or by other aspect of the metabolic disorders associated to diabetes mellitus, such as hypercholesterolemia, the next experiments were performed in the apoE-null mice,26Candido R Allen TJ Lassila M Cao Z Thallas V Cooper ME Jandeleit-Dahm KA Irbesartan but not amlodipine suppresses diabetes-associated atherosclerosis.Circulation. 2004; 109: 1536-1542Crossref PubMed Scopus (191) Google Scholar which are characterized by elevated levels of total serum cholesterol (mean ± SD, 14.27 ± 2.1 mmol/L) with respect to littermate mice (mean ± SD, 1.81 ± 0.42 mmol/L). In these animals, STZ-induced diabetes
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