Osteoprotegerin Group Research Articles

Association between osteoprotegerin, fetuin-A, carotid intima media thickness, and urinary albumin excretion in Type 2 diabetes

Vascular calcification and atherosclerosis play a vital role in the development of cardiovascular morbidity and mortality in diabetic patients, especially when complications of diabetic nephropathy occur. Osteoprotegerin (OPG) and fetuin-A are two markers of vascular calcification. We evaluated the association between these vascular markers and urinary albumin excretion in diabetic patients. Three groups were arranged containing 40 patients: normoalbuminuric (Group 1), microalbuminuric (Group 2), and macroalbuminuric (Group 3). In addition to the obtained data, levels of hs-CRP (high sensitivity-CRP) and homocysteine were examined. OPG levels of patients in Group 2 were higher than in Group 1 (p = 0.058). OPG levels in Group 3 were lower than in Groups 1 or 2 (p = 0.014 and 0.000, respectively). Levels of fetuin-A in Group 2 were determined to be lower than in Groups 1 and 3 (p = 0.001 and 0.000, respectively). Carotid intima media thickness (CIMT) in Group 3 was higher than in Group 1 (p = 0.002). CIMT in Group 2 was also higher than in Group 1 (p = 0.039). A positive correlation between fetuin-A and OPG was found (p = 0.012, r = 0.393). Additionally, a positive correlation between hs-CRP and fetuin-A in Group 2 (p = 0.020, r = 0.367) and a negative correlation between hs-CRP and OPG in Group 3 (p = 0.036, r = -0.333) were observed. The differences found between albuminuria and OPG or fetuin-A may be due to the different doses and variety of medications the patients received, in addition to genetic and racial factors. So far, in our country, polymorphisms related to OPG and fetuin-A have not been defined. Further detailed studies about polymorphisms will have additional value.

OPG inhibits gene expression of RANK and CAII in mouse osteoclast-like cell

This study was designed to determine the effects of the osteoprotegerin (OPG) on the mRNA expression of carbonic anhydrase II (CAII) and the receptor activator of NF-κB (RANK) in mouse osteoclast-like cells. Marrow cells were harvested from femora and tibiae of mouse and cultured in 6-well chamber slides. After 1 day of incubation, the marrow cells were exposed to M-CSF (25 ng/ml), RANKL (50 ng/ml), and different concentrations of OPG (50, 75, and 100 ng/ml, respectively) for 3 days. Osteoclast-like cells were confirmed by both tartrate-resistant acid phosphatase (TRAP) stain and bone resorption assay. The expression of RANK and CAIImRNA was determined with real-time fluorescent quantitative polymerase chain reaction. The numbers of multinucleated, TRAP-positive osteoclast-like cells, and resorption pits formed were observed. Compared with the M-CSF + RANKL group, RANKmRNA expression was statistically decreased in the M-CSF and M-CSF + RANKL + OPG (100 ng/ml) groups (P = 0.004, P = 0.024, respectively); Compared with the M-CSF, M-CSF + RANKL, and M-CSF + RANKL + OPG (100 ng/ml) group, CAIImRNA expression in the M-CSF + RANKL + OPG (75 ng/ml) groups was statistically decreased (P = 0.001, P = 0.008, and P = 0.036, respectively). These data suggest that OPG could regulate the expression of RANK and CA II mRNA in the marrow culture system.

Effects of osteoprotegerin from transfection of pcDNA3.1(+)/chOPG on bioactivity of chicken osteoclasts

BackgroundOsteoprotegerin (OPG) has been reported to prevent bone resorption by inhibiting the formation, function, and survival of osteoclasts in a variety of animal models. However, the effects of OPG on bone metabolism in avian species have not been described. The objective of this study was to investigate the effects of chicken OPG (chOPG) expressed in chicken embryo fibroblasts (CEFs) on chicken osteoclast function in vitro.MethodsThe chOPG sequence containing the open reading frame (ORF) was amplified from chicken embryo frontal bone and inserted into the pcDNA3.1 (+) vector. PcDNA3.1 (+)/chOPG was transiently transfected into CEFs by lipofectamine 2000. Transcription of OPG mRNA and expression of chOPG recombinant protein were detected by reverse transcription polymerase chain reaction (RT-PCR) and indirect immunofluorescence. The level of chOPG recombinant protein was detected by enzyme-linked immunosorbent assay. The suspension of osteoclasts was separated from chicken embryos and divided into three groups (control group, pcDNA3.1 (+) group and pcDNA3.1 (+)/chOPG group). The percentage of osteoclast apoptosis was detected by flow cytometry. The tartrate-resistant acid phosphatase (TRAP) secreted by osteoclasts was measured by the diazol method. The resorbing activity of osteoclasts was evaluated by the area of lacunae on bone flaps and the concentration of calcium in the supernatant liquid of osteoclasts.Results48 h after transfection, the exogenous OPG gene transcription was detected by RT-PCR. After 72 h, the CEFs transfected from pcDNA3.1 (+)/chOPG displayed green fluorescence and the concentration of chOPG protein was 15.78 ± 0.22 ng/mL. After chicken osteoclasts were cultured for 5 d in a medium containing supernatant from transfected CEFs, the percentage of osteoclast apoptosis was increased significantly, the concentration of TRAP, the area of lacunae on bone flaps and calcium concentration were decreased significantly in the pcDNA3.1(+)/OPG group compared to the control group and the pcDNA3.1 (+) group.ConclusionConstructed pcDNA3.1 (+)/chOPG transfected into CEFs expressed bioactive OPG protein that was able to inhibit osteoclast function.

The effect of osteoprotegerin gene modification on wear debris-induced osteolysis in a murine model of knee prosthesis failure

Using an in vivo adeno-associated virus (AAV)-mediated gene transfer technique, this study evaluated the therapeutic effects of an osteoprotegerin (OPG) transgene against orthopaedic wear debris-induced osteolysis in a long-term murine model. A titanium pin was surgically implanted into proximal tibia of Balb/c mice to mimic a weight-bearing knee arthroplasty, followed by an intra-articular challenge with Ti particles to provoke periprosthetic inflammation and osteolysis. rAAV-hOPG or AAV-LacZ vectors were injected into the prosthetic joint at 3 weeks post-op. The tissues were harvested at 2, 4, 12 and 24 weeks after transduction for histological and molecular analyses. Successful transgene expression at the local site was confirmed by real-time PCR and ELISA. Inflammatory pseudo-membranes were ubiquitously present at the interface between the Ti implant and the surrounding bone in both LacZ and virus-free control groups, while soft tissue was only observed sporadically at the bone–implant interface in the OPG group. A significant reduction in TRAP+ osteoclast numbers was observed in the OPG treatment group. MicroCT assessment indicated a marked reversal in the loss of peri-implant bone mineral density (BMD) in the OPG-transduced group, when compared with the LacZ and virus-free controls. Further, OPG gene modification appeared to reduce local bone collagen loss by a mean of 40%. Real-time PCR examination confirmed that in vivo OPG gene transfer dramatically influenced the periprosthetic tissue gene expression profiles by diminishing the mRNA expression of TNF, IL-1, CPK and RANKL. There were no transgene-associated toxic effects apparent during the experiment, and the PCR detection of transgenes in remote organs such as lungs, kidneys, liver, and muscle of contralateral limb were consistently negative. Overall, rAAV-mediated OPG gene transfer effectively reversed Ti-particle-induced bone resorption in this experimental model. The therapeutic effects may be due to the blockage of local osteoclastogenesis and possibly the down-regulation of RANKL expression.

Elevated Osteoprotegerin Levels Predict Cardiovascular Events in New Hemodialysis Patients

Background: Patients on hemodialysis (HD) frequently experience cardiovascular events associated with vascular calcification. We investigated the involvement of osteoprotegerin (OPG), an inhibitor of vascular calcification, in the incidence of cardiovascular events and mortality among new HD patients. Methods: We conducted a prospective cohort study of the association of serum OPG levels with morbidity and mortality in subjects who became new HD patients between June 2000 and May 2006. Results: A total of 99 patients (age 58.9 ± 14.6 years, 65 male, 34 female) were prospectively followed up for 41.5 ± 20.2 months. During this period, 27 patients developed cardiovascular events and 12 died of causes related to cardiovascular disease. When divided into 2 groups according to OPG levels, the high OPG group showed a higher prevalence of cardiovascular morbidity and mortality compared with the low OPG group. Cox’s proportional hazards analysis associated the new onset of cardiovascular events with the high OPG group (HR 2.88, 95% CI 1.09–7.62, p = 0.033). Furthermore, the high OPG group at the start of HD was significantly associated with older age, male gender and a high aortic calcification index. Conclusions: Elevated levels of serum OPG in new HD patients may predict subsequent cardiovascular events.

The Effect of Osteoclastic Activity on Tendon-to-Bone Healing

Healing of a tendon graft in a bone tunnel depends on bone ingrowth into the interface between tendon and bone. Excessive osteoclastic activity may contribute to bone resorption, tunnel widening, and impaired healing. We hypothesized that inhibition of osteoclastic activity by osteoprotegerin (OPG) would increase bone formation around a tendon graft in anterior cruciate ligament reconstruction in a rabbit model, while increased osteoclastic activity due to the application of receptor activator of nuclear factor-kappa B ligand (RANKL) would impair bone ingrowth. Sixty skeletally mature, male New Zealand White rabbits underwent bilateral anterior cruciate ligament reconstruction. OPG (100 microg per tunnel) or RANKL (10 microg per tunnel) was delivered to the tendon-bone interface with use of a synthetic calcium phosphate carrier vehicle. Twenty animals were killed at two, four, and eight weeks after surgery. Two rabbits from each group were prepared for histological evaluation, and the other rabbits were used for biomechanical testing. A significantly greater amount of bone surrounded the tendon at the healing tendon-bone interface in the OPG-treated limbs compared with the controls and the RANKL-treated limbs at all time-points (p < 0.05). There were significantly fewer osteoclasts in the OPG-treated limbs compared with the controls and the RANKL-treated limbs (p < 0.05). The average tunnel area in the OPG group was significantly smaller than that in the RANKL group (p = 0.003 at two weeks and p = 0.004 at four weeks). The femur-anterior cruciate ligament-tibia complex of the OPG-treated limbs had significantly increased stiffness compared with RANKL-treated limbs at eight weeks (p = 0.04). Osteoprotegerin significantly improves bone formation around the grafted tendon and improves the stiffness at the healing tendon-bone junction in a rabbit model.

The Effect of Osteoclastic Activity on Tendon-to-Bone Healing

Background: Healing of a tendon graft in a bone tunnel depends on bone ingrowth into the interface between tendon and bone. Excessive osteoclastic activity may contribute to bone resorption, tunnel widening, and impaired healing. We hypothesized that inhibition of osteoclastic activity by osteoprotegerin (OPG) would increase bone formation around a tendon graft in anterior cruciate ligament reconstruction in a rabbit model, while increased osteoclastic activity due to the aplication of receptor activator of nuclear factor-kappa B ligand (RANKL) would impair bone ingrowth. Methods: Sixty skeletally mature, male New Zealand White rabbits underwent bilateral anterior cruciate ligament reconstruction. OPG (100 μg per tunnel) or RANKL (10 μg per tunnel) was delivered to the tendon-bone interface with use of a synthetic calcium phosphate carrier vehicle. Twenty animals were killed at two, four, and eight weeks after surgery. Two rabbits from each group were prepared for histological evaluation, and the other rabbits were used for biomechanical testing. Results: A significantly greater amount of bone surrounded the tendon at the healing tendon-bone interface in the OPG-treated limbs compared with the controls and the RANKL-treated limbs at all time-points (p < 0.05). There were significantly fewer osteoclasts in the OPG-treated limbs compared with the controls and the RANKL-treated limbs (p < 0.05). The average tunnel area in the OPG group was significantly smaller than that in the RANKL group (p = 0.003 at two weeks and p = 0.004 at four weeks). The femur-anterior cruciate ligament-tibia complex of the OPG-treated limbs had significantly increased stiffness compared with RANKL-treated limbs at eight weeks (p = 0.04). Conclusions: Osteoprotegerin significantly improves bone formation around the grafted tendon and improves the stiffness at the healing tendon-bone junction in a rabbit model. Clinical Relevance: Inhibition of excessive osteoclastic activity may improve tendon-to-bone healing.

347 POSTER The RANK ligand inhibitor OPG-Fc reduces bone lesions and skeletal tumor burden in the MDA-231 breast cancer and PC3 prostate cancer experimental osteolytic metastases models

Recombinant human BMP-2 (rhBMP-2) is a potent osteoinductive agent, but has been associated not only with bone formation, but also osteoclastogenesis and bone resorption. Osteoprotegerin (OPG) is a RANKL inhibitor that blocks differentiation and function of osteoclasts. We hypothesized that the combination of local BMP-2 (recombinant protein or a product of gene therapy) plus systemic OPG-Fc is more effective than BMP-2 alone in promoting bone repair. To test this hypothesis we used a mouse critical-sized femoral defect model. Col2.3eGFP (osteoblastic marker) male mice were treated with rhBMP-2 (group I), rhBMP-2 and systemic OPG (group II), rhBMP-2 and delayed administration of OPG (group III), mouse BM cells transduced with a lentiviral vector containing the BMP-2 gene (LV-BMP-2; group IV), LV-BMP-2 and systemic OPG (group V), a carrier alone (group VI) and administration of OPG alone (group VII). All bone defects treated with BMP-2 (alone or combined with OPG) healed, whereas minimal bone formation was noted in animals treated with the carrier alone or OPG alone. MicroCT analysis showed that bone volume (BV) in rhBMP-2 + OPG and LV-BMP-2 + OPG groups was significantly higher compared to rhBMP-2 alone (p < 0.01) and LV-BMP-2 alone (p < 0.001). Similar results were observed in histomorphometry, with rhBMP-2 alone defects exhibiting significantly lower bone area (B.Ar) compared to rhBMP-2 + OPG defects (p < 0.005) and LV-BMP-2 defects having a significantly lower B.Ar compared to all BMP-2 + OPG treated groups (p ≤ 0.01). TRAP staining demonstrated a major osteoclast response in the groups that did not receive OPG (rhBMP-2, LV-BMP-2 and sponge alone) beginning as early as 7 days post-operatively. In conclusion, we demonstrated that locally delivered BMP-2 (recombinant protein or gene therapy) in combination with systemically administered OPG improved bone healing compared to BMP-2 alone in a mouse critical-sized bone defect. These data indicate that osteoclasts can diminish healing responses to BMP-2 and that RANKL inhibition may thus accentuate BMP-2 efficacy.

Osteoprotegerin and Bone Mineral Density in Hemodiafiltration Patients

A newly identified cytokine, osteoprotegerin (OPG) appears to be involved in the regulation of bone remodeling. In vitro studies suggest that OPG, a soluble member of the TNF receptor family of proteins, inhibits osteoclastogenesis by interrupting the intercellular signaling between osteoblastic stromal cells and osteoclast progenitors. As patients with chronic renal failure (CRF) often have renal osteodystrophy (ROD), we investigated the role of osteoprotegerin (OPG) in ROD, and investigated whether there was any relationship between serum OPG, intact parathyroid (PTH) (iPTH), vitamin D, and trabecular bone. Serum OPG combined with iPTH might be a useful tool in the noninvasive diagnosis of ROD, at least in cases in which the range of PTH values compromises reliable diagnosis. Thirty-six patients on maintenance hemodiafiltration (HDF) and a control group of 36 age and sex matched healthy subjects with no known metabolic bone disease were studied. The following assays were made on serum: iPTH, osteocalcin (BGP), bone alkaline phosphatase, 25(OH)-cholecalciferol, calcium, phosphate, OPG, IGF-1, estradiol, and free testosterone. Serum Ca+ +, P, B-ALP, BGP, IGF-1, iPTH, and OPG levels were significantly higher in HDF patients than in controls, while DXA measurements and quantitative ultrasound (QUS) parameters were significantly lower. On grouping patients according to their mean OPG levels, we observed significantly lower serum IGF-1, vitamin D3 concentrations, and lumbar spine and hip bone mineral density in the high OPG groups. No correlation was found between OPG and bone turnover markers, whereas a negative correlation was found between serum OPG and IGF-1 levels (r= − 0.64, p = 0.032). Serum iPTH concentrations were positively correlated with bone alkaline phosphatase (B-ALP) (r = 0.69, p = 0.038) and BGP (r = 0.92, p < 0.001). The findings made suggest that an increase in OPG levels may be a compensatory response to elevated bone loss. The low bone mineral density (BMD) levels found in the high OPG group might have been due to the significant decrease in serum IGF-1 and vitamin D3 observed. In conclusion, the findings made in the present study demonstrate that increased OPG in hemodiafiltration patients is only partly due to decreased renal clearance. As it may partly reflect a compensatory response to increased bone loss, this parameter might be helpful in the identification of patients with a marked reduction in trabecular BMD.