RESUMEN
UNLABELLED: Bisphosphonates (BPs) are widely used in the treatment of a variety of bone-related diseases, particularly where the bone turnover is skewed in favor of osteolysis. The mechanisms by which BPs reduce bone resorption directly acting on osteoclasts are now largely clarified even at molecular level. Researches concerning the BP's effects on osteoblast have instead shown variable results. Many in vitro studies have reported positive effects on osteoblasts proliferation and mineralization for several BPs; however, the observed effects differ, depending on the variety of different model system that has been used. OBJECTIVES: We have investigated if neridronate, an aminobisphosphonate suitable for pulsatory parenteral administration, could have an effect on human osteoblastic proliferation and differentiation in vitro. METHODS: We have investigated whether prolonged addition of neridronate (from 10(-3) to 10(-11) M) to different human osteoblasts cultures, obtained from 14 different bone specimens, could affect the cells number, the endogenous cellular alkaline phosphatase (ALKP) activity, and the formation of mineralized nodules. RESULTS: Our results show that neridronate does not negatively affect in vitro the viability, proliferation, and cellular activity of normal human osteoblasts even after a long period addition of the drug (20 days) at concentrations equal or lower than 10(-5) mol/l (therapeutic dose). In addition, neridronate seems to enhance the differentiation of cultured osteoblasts in mature bone-forming cells. A maximum increase of alkaline phosphatase activity (+50% after 10 days; P < 0.01) and mineralized nodules (+48% after 20 days; P < 0.05) was observed in cultures treated with neridronate 10(-8) M. CONCLUSIONS: These results encourage the use of neridronate in long-term therapy of demineralizing metabolic bone disorders.
Asunto(s)
Difosfonatos/farmacología , Osteoblastos/citología , Osteoblastos/efectos de los fármacos , Anciano , Fosfatasa Alcalina/metabolismo , Calcificación Fisiológica/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Osteoblastos/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de TiempoRESUMEN
OBJECTIVE: Experimental and clinical data suggest that statins may protect bone by inhibiting bone resorption and/or stimulating bone formation. Interleukin-6 (IL-6) is produced by osteoblasts, and potently stimulates osteoclast activation playing a key role in normal bone resorption as well as in post-menopausal and inflammation-driven osteoporosis. Although statins inhibit IL-6 production from different cell types, currently no data exist on osteoblasts. The aim of the study was to evaluate the effect of rosuvastatin on IL-6 production by human osteoblasts. METHODS: Osteoblasts from osteoarthritic patients were incubated with rosuvastatin (0.1-10 µmol/L)±IL-1ß, and IL-6 production was evaluated as cytokine concentration in the culture medium (ELISA), as well as mRNA expression in the cells (qPCR). Putative intracellular mechanisms of the drug, such as blocking HMG-CoA-reductase, and interference in the prenylation process were investigated by the addition of mevalonate and isoprenoids. The effect of rosuvastatin±IL-1ß on the anti-resorptive molecule osteoprotegerin (OPG) was also assessed (ELISA). RESULTS: Rosuvastatin significantly reduced IL-6 levels in the osteoblast culture medium, both in unstimulated and IL-1ß-stimulated cells. This effect was reversed by mevalonate or geranylgeraniol, but not farnesol. Moreover, the drug decreased both spontaneous and IL-1ß-induced IL-6 mRNA expression in osteoblasts. Conversely, rosuvastatin did not affect OPG levels in the culture medium. CONCLUSION: Our results show that rosuvastatin decreases IL-6 production by osteoblasts, thereby suggesting a possible inhibiting activity on osteoclast function in an indirect way. These data may provide further rationale for employing rosuvastatin to beneficially affect bone metabolism in post-menopausal women and possibly in inflammation-driven osteoporosis.
Asunto(s)
Fluorobencenos/farmacología , Interleucina-1beta/farmacología , Interleucina-6/genética , Osteoblastos/efectos de los fármacos , Osteoblastos/fisiología , Pirimidinas/farmacología , Sulfonamidas/farmacología , Anciano , Resorción Ósea/tratamiento farmacológico , Resorción Ósea/metabolismo , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Medios de Cultivo/farmacología , Diterpenos/farmacología , Farnesol/farmacología , Cabeza Femoral/citología , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/farmacología , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Interleucina-6/metabolismo , Ácido Mevalónico/farmacología , Persona de Mediana Edad , Osteoblastos/citología , Osteoporosis/tratamiento farmacológico , Osteoporosis/metabolismo , Osteoprotegerina/metabolismo , ARN Mensajero/metabolismo , Rosuvastatina Cálcica , Terpenos/farmacologíaRESUMEN
Vascular endothelial growth factor (VEGF)-D is a member of the VEGF family of angiogenic growth factors that recognizes and activates the vascular endothelial growth factor receptor (VEGFR)-2 and VEGFR-3 on blood and/or lymphatic vessels. We show that in the long bones of newborn mice, VEGF-D and VEGFR-3 are expressed in the osteoblasts of the growing plate. The treatment of primary human osteoblasts with recombinant VEGF-D induces the expression of osteocalcin and the formation of mineralized nodules in a dose-dependent manner. A monoclonal neutralizing antibody, anti-VEGF-D, or silencing of VEGFR-3 by lentiviral-mediated expression of VEGFR-3 small hairpin RNA affects VEGF-D-dependent osteocalcin expression and nodule formation. Moreover, in primary human osteoblasts, VEGF-D expression is under the control of VEGF, and inhibition of VEGF-D/VEGFR-3 signaling, by monoclonal antibodies or VEGFR-3 silencing, affects VEGF-dependent osteoblast differentiation. These experiments establish that VEGF-D/VEGFR-3 signaling plays a critical role in osteoblast maturation and suggest that VEGF-D is a downstream effector of VEGF in osteogenesis.
Asunto(s)
Osteoblastos/citología , Osteoblastos/fisiología , Osteogénesis/fisiología , Factor D de Crecimiento Endotelial Vascular/metabolismo , Receptor 3 de Factores de Crecimiento Endotelial Vascular/metabolismo , Anciano , Animales , Animales Recién Nacidos , Diferenciación Celular/fisiología , Células Cultivadas , Endotelio Vascular/citología , Femenino , Humanos , Masculino , Ratones , Persona de Mediana Edad , Osteocalcina/genética , ARN Interferente Pequeño , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/farmacología , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Venas Umbilicales/citología , Factor D de Crecimiento Endotelial Vascular/genética , Factor D de Crecimiento Endotelial Vascular/farmacología , Receptor 3 de Factores de Crecimiento Endotelial Vascular/genéticaRESUMEN
Two poly(amido-amine)s (oligoPAM and oligoNER) containing bis-phosphonate residues were obtained by a Michael-type polyaddition of pamidronate and neridronate to 1,4-bis(acryloyl)piperazine. The SEC (size-exclusion chromatography) and the MALDI-TOF (matrix assisted laser desorption ionization) analyses were consistent with the presence of oligomeric species (2-3 kDa) and with a narrow polydispersity index. The thermodynamic results (log Ks, -DeltaH(o) , and DeltaS(o) obtained at 25 degrees C in 0.15 M NaCl) of both the oligomers and the corresponding low molecular weight precursors were in line with a cluster structure formed during the protonation of the basic nitrogen in the pamidronate. The solubility of the oligoNER with a longer aliphatic chain was improved at high pHs, allowing the evaluation of their solution properties. Preliminary biological results show that both the oligomers do not negatively affect the in vitro viability, proliferation, and cellular activity of either normal animal or human osteoblasts.
Asunto(s)
Resorción Ósea/tratamiento farmacológico , Difosfonatos/química , Difosfonatos/uso terapéutico , Supervivencia Celular/efectos de los fármacos , Difosfonatos/toxicidad , Diseño de Fármacos , Humanos , Modelos Moleculares , Pamidronato , Piperazinas , Potenciometría , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Termodinámica , ViscosidadRESUMEN
Changes in expression profiles for 17 proteins were ascertained in human mature osteoblasts compared to pre-osteoblasts (differentiation markers). A differential approach was used to highlight proteomic changes between human osteosarcoma cells and mature osteoblasts, showing a relative over-expression of 8 proteins (proliferation and tumor indicators), as well as under-expression of proteins also found down-regulated in pre-osteoblasts (specific markers of osteoblast differentiation). Our findings confirmed the differences between cell lines and primary human cell cultures and suggested caution on the use of osteosarcoma to study anti-osteoporotic drugs in humans.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Osteoblastos/fisiología , Proteoma/análisis , Proteómica/métodos , Adulto , Fosfatasa Alcalina/análisis , Biomarcadores de Tumor/análisis , Huesos/citología , Técnicas de Cultivo de Célula , Línea Celular Tumoral , Células Cultivadas , Subunidad alfa 1 del Factor de Unión al Sitio Principal/metabolismo , Regulación de la Expresión Génica , Humanos , Masculino , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoblastos/ultraestructura , Osteocalcina/metabolismo , Osteosarcoma/patologíaRESUMEN
The osteogenic growth peptide (OGP) is a naturally occurring tetradecapeptide that has attracted considerable clinical interest as a bone anabolic agent and hematopoietic stimulator. In vivo studies on animals have demonstrated that the synthetic peptide OGP (10-14), reproducing the OGP C-terminal active portion [H-Tyr-Gly-Phe-Gly-Gly-OH] increases bone formation, trabecular bone density and fracture healing. In vitro studies performed on cellular systems based on osteoblastic-like cell lines or mouse stromal cells, have demonstrated that OGP (10-14) increases osteoblast proliferation, alkaline phosphatase (ALKP) activity and matrix synthesis and mineralization. In view of a potential application of OGP (10-14) in clinical therapy, we have tested different concentrations of OGP (10-14) on primary human osteoblast (hOB) cultures. We have observed significant increases of hOB proliferation (+35%), ALKP activity (+60%), osteocalcin secretion (+50%), and mineralized nodules formation (+49%). Our experimental model based on mature hOBs was used to investigate if OGP (10-14) could prevent the effects on bone loss induced by sustained glucocorticoid (GC) treatments. A strong decrease in bone formation has been attributed to the effects of GCs on osteoblastogenesis and osteocyte apoptosis, while an increase in bone resorption was due to a transient osteoblastic stimulation, mediated by the OPG/RANKL/RANK system, of osteoclasts recruitment and activation. Moreover, GCs act on hOBs decreasing the release of osteoprotegerin (OPG) a regulator of the RANKL/RANK interaction. Here, we provide evidences that OGP (10-14) inhibits hOB apoptosis induced by an excess of dexamethasone (-48% of apoptotic cells). Furthermore, we show that OGP (10-14) can increase OPG secretion (+20%) and can restore the altered expression of OPG induced by GCs to physiological levels. Our results support the employment of OGP (10-14) in clinical trials addressed to the treatment of different bone remodeling alterations including the GC-induced osteoporosis.