LIOMM
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El Laboratorio de InvestigaciĆ³n en OsteopatĆas y Metabolismo Mineral fue creado en el aƱo 2012 como una unidad multidisciplinaria dedicada a la investigaciĆ³n cientĆfico-tecnolĆ³gica, con el fin de incrementar los conocimientos cientĆficos, la educaciĆ³n y la extensiĆ³n en el campo de las patologĆas Ć³seas y metabĆ³licas, asĆ como su aplicaciĆ³n en la ingenierĆa de tejidos. Nuestras Ć”reas de interĆ©s son: OsteopatĆas, Metabolismo Mineral, Diabetes mellitus, SĆndrome MetabĆ³lico, IngenierĆa de Tejido. Abordamos aspectos de la fisiopatologĆa del esqueleto, asociadas con enfermedades metabĆ³licas de alta prevalencia como la Diabetes mellitus, SĆndrome MetabĆ³lico y obesidad. Investigamos las posibles causas de estas osteopatĆas metabĆ³licas, sus tratamientos con diferentes fĆ”rmacos, asĆ como terapia celular usando cĆ©lulas progenitoras de mĆ©dula Ć³sea. Para contribuir al espectro terapĆ©utico disponible para las distintas patologĆas Ć³seo-cartilaginosas, desarrollamos y estudiamos matrices polimĆ©ricas que sirvan como sistemas de liberaciĆ³n controlada de drogas, y/o como scaffolds para la reparaciĆ³n del tejido oseo-articular.
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Embargado Multiple modulators of the glucose-induced net calcium uptake by isolated islets(1984) Borelli, MarĆa InĆ©s; Cortizo, Ana MarĆa; Gagliardino, Elma Edith P. de; Garcia, MarĆa Elisa; Gagliardino, Juan JosĆ©Glucose-induced insulin secretion and net calcium uptake were simultaneously studied in isolated islets obtained from normal, adrenalectomized, ovariectomized and radiothyroidectomized rats, as well as from the corresponding hormone deprived rats following the administration of specific substitutive therapy. Both parameters were also studied in islets from normal rats incubated in the presence of Trifluoperazine (TFP). In all these unrelated experimental conditions simultaneous changes were obtained, observed in the release of insulin and the net calcium uptake elicited by glucose. Otherwise, the modifications of these two parameters obtained in the hormone deprived states were brought back to normal when the animals received the specific substitutive hormonal treatment. On the other hand, TFP also induces simultaneous diminution in both glucose-induced insulin release and net calcium uptake by isolated islets. On account of our results, we could suggest that the mechanism involved in the control of the glucose-induced net calcium uptake is actively modulated by adrenal and ovarian steroids and thyroid hormones as well as by calmodulin. Therefore, changes induced either in the level or activity of these modulators will modify the rate of influx and efflux of Ca2+ across the plasma membrane, with the consequent alteration in the mechanism of stimulus: secretion coupling of insulin. - Comunicacion
Acceso Abierto Effect of hypothyroidism on the composition and turnover rate of islet phospholipids(1984) Cortizo, Ana MarĆa; Garcia, M.E.; Pasquini, J.M.; Gagliardino, J.It has already been demonstrated that the pancreatic B cells of hypothyroid rats have a reduced capacity to release insulin in response to glucose (l). This impaired B cell function may be partly due to a diminished rate of glucose oxidation and net calcium uptake associated with ultrastructural alteration of the pancreatic islets (2). To identify further other factors responsible for this diminished B cell secretory function, we studied the composition and the turnover rate of phospholipids in islets obtained from hypothyroid rats. - ArtĆculo
Acceso Abierto Mecanismo de acciĆ³n de hormonas tiroideas sobre el pĆ”ncreas endĆ³crino(1987) Cortizo, Ana MarĆaEl rol de las hormonas tiroideas en la Diabetes Mellitus y la funciĆ³n endocrina del pĆ”ncreas fue estudiado por primera vez en 1944 por B. A. Houssay (1 ). Trabajando con perros parcialmente panceatectomizados, sometidos a tratamiento con hormonas tiroideas, observĆ³ que adquirĆan una forma de diabetes irreversible, que persistĆa aĆŗn despuĆ©s de interrumpir el tratamiento tiroideo. LlamĆ³ a Ć©sta Diabetes Meta.tiroidea ; su mecanismo parecĆa ser un agotamiento pancreĆ”tico consecutivo a una sobreestimulaciĆ³n (2). Sus estudios se extendieron en animales sometidos a tiroidectomĆa y pancreatectomĆa subtotal simultĆ”nea, observando que no se reducĆa la severidad de la diabetes, sino que la operaciĆ³n exacerbaba los sĆntomas, la enfermedad progresaba mĆ”s rĆ”pidamente y finalmente morĆan en hiperglucemia. A partir de estos experimentos, las hormonas tiroideas han sido consideradas como hormonas diabetogĆ©nicas. Numerosos investigadores han observado la intolerancia a la glucosa asociada con una disfunciĆ³n tiroidea, tanto a nivel clĆnico como experimental (3-11). Pero el mecanismo por el cual las hormonas tiroideas llevan a cabo estos cambios es hasta hoy desconocido. - ArtĆculo
Acceso Abierto Changes induced by glucose in the plasma membrane properties of pancreatic islets(1990) Cortizo, Ana MarĆa; Paladini, A.; DĆaz, G.B.; GarcĆa, M.E.; Gagliardino, J.J.Partially purified membranes obtained from rat pancreatic isolated islets preincubated for 3 min with 3.3 and 16.6 mM glucose were labelled with 1,6-diphenyl-1,3,5-hexatriene to study fluorescence polarization. Other islets, incubated for 5 min with the same glucose concentration, were extracted and phospholipids separated by thin-layer chromatography. The composition of phospholipids of fatty acids was then studied by gas-liquid chromatography. Arrhenius plots of the microviscosity in membranes obtained from islets exhibited two components, a steeper slope below 18 degrees C and a gentler slope above 18 degrees C, indicating greater flow activation energy at temperatures below the transition point. Exposure of islets to 16.6 mM glucose significantly increased the flow activation energy (delta E), below and above the transition point. Islets incubated for 5 min with 16.6 mM glucose showed an increase in the percentage composition of 12:0 and 18:2 together with a decrease in the 20:2 W6 and 22:3 W3 fatty acids esterified to phospholipids. Regardless of these changes, no significant alterations occurred in the proportion of saturated fatty acids or in the double bond index; these measurements therefore did not account for the effects of glucose concentration in flow activation energy. The thermotropic changes reported here might be the consequence of some degree of disorder induced by glucose upon the membrane structure. This order alteration could either favor the membrane fusion which occurs during the emiocytosis or only reflects the consequence of such a process. - ArtĆculo
Acceso Abierto Vectorial insulin secretion by pancreatic Ī²-cells(1990) Cortizo, Ana MarĆa; Espinal, Joseph; Hammonds, PeterMorphological studies of pancreatic Ī²-cells have suggested the presence of discrete sensory and secretory domains. In the present study we now provide functional evidence by demonstrating polarity of insulin release by HIT-T15 cells. A significant diffusion barrier across a twin chamber culture system was verified in the presence of confluent HIT-T15 cells. When stimulated with sulphonylurea, ionophore or high potassium, insulin was preferentially released into the lower chamber irrespective of whether secretagogues were added to the upper or lower chambers. Vectorial insulin secretion may be a significant determinant of islet hormone paracrine interactions in the maintenance of glucose homeostasis. - ArtĆculo
Acceso Abierto Stimulated release of arachidonate and prostaglandins is vectorial in MDCK epithelial cells(1992) Cortizo, Ana MarĆa; Besterman, J.M.; Leitner, P.P.; Chandrabose, K.A.The receptor mediated activation of phospholipase A2by appropriate ligands results in the synthesis and release of eicosanoids, a class of potent bioregulatory molecules. Madin-Darby canine kidney cells (MDCK) are polarized epithelial cells, with structurally and functionally distinct plasma membrane domains separated by tight junctions. Using MDCK cells grown in dual sided chambers, we show in this report, that a) the receptor mediated release of prostaglandins and arachidonate into the extracellular medium is predominantly unidirectional, b) the direction of release is agonist specific, and c) the magnitude of the response due to a given agonist is cell-domain specific. These characteristics, if operativein vivo, would contribute towards the optimal function of trans-cellular metabolism of eicosanoids already demonstrated. - ArtĆculo
Acceso Abierto Comparative Study of IGFBP Properties in Toad and Rat Sera(1993) Cortizo, Ana MarĆa; Braziunas, D.; Jasper, H.; Gagliardino, J.J.The levels ofIGF-Ihave been simultaneously measured byradioimmunoassayin samples of the toadBufo arenarumand of normal male Wistar rats. In addition, the different fractions of IGF-I binding proteins (IGFBP) and their binding properties have been identified by ligand blot and Scatchard analysis in the serum of both species. In the toad, we have measured levels of IGF-I (2.78 Ā± 0.48 ng/ml) similar to those previously reported in amphibians but far below those found in rats. IGFBP levels were estimated at 129 Ā± 23 and 4249 Ā± 321 pg/ml in toad and rat serum samples. Two main IGFBP fractions of 30-34 kDa, accompanied by a minor component of 24 kDa and seldom by another of 40 kDa, were identified in toad serum. In rat serumāas already reportedāthree bands of 40, 30, and 24 kDa were identified, the first being the main component and the last the minor one. The Scatchard analysis of a competitive binding assay showed two types of binding sites in toad serum: one of high affinity-low capacity (Ka1= 1.6 Ć 1010M-1;R1= 1.2 Ć 10-11M) and another with low affinity-high capacity (Ka2= 1.9 Ć 108M-1;R2= 1.9 Ć 10-10M). The percentage fraction of these binding sites occupied by IGF-I was 13.5%. The figures for K1and K2were lower and those for R1and R2were higher in rat than in toad serum. The percentage fraction of occupied ratIGFbinding sites was 3.6%. The IGF carrier levels (IGFBP5) estimated in our laboratory in samples of rat and toad serum gave figures that were almost 33 times lower in the latter than in the former. Hence, the fraction of free and bound IGF-I in toad and rat blood might be different. Our results provide new evidence of the presence and the properties of IGFBP in amphibians, confirming the wide distribution of this carrier among different species and its possible role as modulator of IGF-I biological effects. - ArtĆculo
Acceso Abierto Vanadium Compounds(1994) Cortizo, Ana MarĆa; Salice C, Viviana; Etcheverry, Susana B.The direct effect of different vanadium compounds upon alkaline phosphatase (ALP) activity was investigated~ Vanadate and vanadyl inhibited both the soluble and particulate ALP activity from UMR.106 cells and front bovine intestinal ALP. We have also shown the inhibition of ALP activity in the soluble fraction of osteoblasts by peroxo and hydroperoxo vanadium compounds. ALP activity in the particulate fraction was not inhibited by these species; nor was the bovine intestinal ALP. Using inhibitors of Tyr-phosphatase (PTPases), the soluble ALP was partially characterized as a PTPase. The major activity in the particulate fraction represents the bone-specific ALPactivity. This study demonstrates that different forms of vanadium are direct inhibitors of ALP activity. This effect is dependent on the enzymatic activity investigated and on the origin of the ALP. - ArtĆculo
Acceso Abierto Vanadium derivatives act as growth factor--mimetic compounds upon differentiation and proliferation of osteoblast-like UMR106 cells(1995) Cortizo, Ana MarĆa; Etcheverry, Susana B.The effect of different vanadium compounds on proliferation and differentiation was examined in osteoblast-like UMR106 cells. Vanadate increased the cell growth in a biphasic manner, the higher doses inhibiting cell progression. Vanadyl stimulated cell proliferation in a dose-responsive manner. Similar to vanadate, pervanadate increased osteoblast-like cell proliferation in a biphasic manner but no inhibition of growth was observed. Vanadyl and pervanadate were stronger stimulators of cell growth than vanadate. Only vanadate was able to regulate the cell differentiation as measured by cell alkaline phosphatase activity. These results suggest that vanadium derivatives behave like growth factors on osteoblast-like cells and are potential pharmacological tools in the control of cell growth. - ArtĆculo
Acceso Abierto Insulin-like growth factor binding proteins from adult-hamster pancreatic islets: influence of glucose concentration(1997) Massa, L.; Cortizo, Ana MarĆa; Gagliardino, Juan JosĆ©This study investigated the effect of glucose on insulin-like growth factor binding proteins (IGFBPs) in islets isolated from pancreas of adult hamsters and compared the response pattern with that of their serum IGFBPs. Serum samples and islets were obtained from adult normal male hamsters, and IGF-binding capacity was measured in aliquots of serum, sonicated islets, or conditioned medium using either 125I-hIGF-I or -II. IGFBPs were characterized in these samples by the ligand-blotting technique, and insulin was measured in conditioned medium by radioimmunoassay. Three IGFBP fractions were identified in serum, with relative molecular weights of 38, 30-33, and 24 kDa, while only two fractions of 30-33 and 24 kDa were identified in islets or in their conditioned medium. Islets cultured with 2 or 16 mM glucose for 48 h released more insulin in the presence of the higher glucose concentration. The binding capacity measured in the islet suspension or conditioned medium increased as a function of glucose concentration in the incubation medium. The IGFBPs present both in islets and conditioned medium had a 3- to 4-fold higher apparent affinity for IGF-II than IGF-I. The higher glucose concentration increased the intensity of the two IGFBP bands identified in the islet suspension by 2- to 3-fold. Our data show that two low-molecular-weight IGFBPs were released from adult hamster pancreatic islets, with a different distribution pattern from that of hamster serum, and that the amount of IGFBPs released by islets depended on the glucose concentration in the culture medium. Though not conclusive, these data suggest that IGFBPs may play a regulatory role in B-cell turnover in adult islets as they do in foetal islets. - ArtĆculo
Acceso Abierto Proliferative and morphological changes induced by vanadium compounds on Swiss 3T3 fibroblasts(1997) Cortizo, Ana MarĆa; Salice C, Viviana; Vescina, Cecilia M.; Etcheverry, Susana B.Vanadium compounds are shown to have a mitogenic effect on fibroblast cells. The effects of vanadate, vanadyl and pervanadate on the proliferation and morphological changes of Swiss 3T3 cells in culture are compared. Vanadium derivatives induced cell proliferation in a biphasic manner, with a toxic-like effect at doses over 50 mM, after 24 h of incubation. Vanadyl and vanadate were equally potent at 2.5ā10 mM. At 50 mM vanadate inhibited cell proliferation, whereas slight inhibition was observed at 100 mM of vanadyl. At 10 mM pervanadate was as potent as vanadate and vanadyl in stimulating fibroblast proliferation, but no effect was observed at lower concentrations. A pronounced cytotoxic-like effect was induced by pervanadate at 50 mM. All of these effects were accompanied by morphological changes: transformation of fibroblast shape from polygonal to fusiform; retraction with cytoplasm condensation; and loss of lamellar processes. The magnitude of these transformations correlates with the potency of vanadium derivatives to induce a cytotoxic-like effect: pervanadate > vanadate > vanadyl. These data suggest that the oxidation state and coordination geometry of vanadium determine the degree of the cytotoxicity - ArtĆculo
Acceso Abierto Relationship between non-enzymatic glycosylation and changes in serum insulin-like growth factor-1 (IGF-1) and IGF-binding protein-3 levels in patients with type 2 diabetes mellitus(1998) Cortizo, Ana MarĆa; Lee, P.D.; CĆ©dola, N. V.; Jasper, H.; Gagliardino, J.J.The possible occurrence of increased non-enzymatic glycosylation of serum insulin-like growth factor binding protein-3 (IGFBP-3) in vivo and the changes that would simultaneously occur in serum levels of IGFBP-3 and insulin-like growth factor-1 (IGF-I) were investigated. We measured levels of IGF-I and IGFBP-3 and the degree of glycation of total serum protein and IGFBP-3, in serum samples obtained from patients with poorly controlled non-insulin-dependent diabetes (type 2) and from age-matched non-diabetic controls. Type 2 diabetic patients had significantly higher glycated serum protein (GlyP) levels. GlyP significantly correlated with age in the control (r = 0.315, P<0.05) but not in the type 2 diabetes group. Control and diabetic subjects had comparable serum IGF-I levels and in both groups IGF-I levels tended to decrease with age (r = -0.567, P<0.001 and r = -0.465, P<0.05 for control and type 2 diabetic subjects, respectively). In the type 2 diabetes group, IGF-I levels showed a negative correlation with serum GlyP values (r = -0.476, P<0.05). Type 2 diabetic and control patients had comparable serum IGFBP-3 levels, which were significantly higher in diabetic patients in the older age subgroups. A negative correlation was found between IGFBP-3 levels and age in the control (r = -0.705, P<0.001) and in the type 2 diabetes groups (r = -0.463, P<0.05). A significant negative correlation was found between IGFBP-3 levels and GlyP in control (r = -0.449, P<0.002) but not in type 2 diabetic subjects. The mean glycated IGFBP-3 (GlyIGFBP-3) levels were higher in the oldest type 2 diabetic patients. In these patients, GlyIGFBP-3 was negatively associated with IGF-I levels (r = -0.447, P<0.05). The IGF-I/IGFBP-3 molar ratio was significantly reduced in the 46-60-year-old type 2 diabetic group, whereas the IGF-I/IGFBP-3 ratio was positively and significantly correlated with GlyP levels only in the control group (r = 0.489, P<0.01). Our results show that: a) increased non-enzymatic glycosylation of IGFBP-3 occurs in vivo; and b) this effect is accompanied by an increase in IGFBP-3 levels. These results suggest that the IGF-I/IGFBP-3 system is another target for the metabolic derangements of type 2 diabetes. Its alterations might play a role in diabetic complications. - ArtĆculo
Acceso Abierto Factores de crecimiento insulino sĆmiles: estructura, bioactividad y mĆ©todos de ensayo(1998) Cortizo, Ana MarĆa; Mccarthy, AntonioEl sistema de los factores de crecimiento insulino-sĆmiles (IGF) se halla involucrado en diferentes aspectos de la regulaciĆ³n celular y tisular, como asĆ tambiĆ©n en el desarrollo y el crecimiento corporal. Este sistema depende de la interacciĆ³n entre ligandos (IGF-I, IGF-II), receptores (Tipo I, Tipo II), proteĆnas ligadoras o transportadoras (IGFBP-1 a -6), y proteasas especĆficas para las IGFBPs. La acciĆ³n de los IGFs se encuentra regulada por diferentes factores y estĆmulos, tales como la hormona de crecimiento, que actĆŗan a diversos niveles. El desarrollo de nuevos mĆ©todos para analizar los diferentes componentes del sistema de los IGFs ha aportado elementos adicionales para la evaluaciĆ³n, diagnĆ³stico y seguimiento de pacientes con alteraciones del crecimiento. - ArtĆculo
Acceso Abierto Vanadate-induced nitric oxide production: role in osteoblast growth and differentiation(2000) Cortizo, Ana MarĆa; Caporossi, Mariana; Lettieri, Gabriela; Etcheverry, Susana B.Nitric oxide NO. has been shown to act as a mediator of cytokines in bone tissue. We have previously demonstrated that vanadium compounds are insulin- and growth factor-mimetic compounds in osteoblasts in culture, although high doses are toxic to these cells. In this study, we measured NO production in two osteoblast-like cells UMR106 and MC3T3E1. incubated with different concentrations 2.5ā100 mM. of vanadate. Vanadate induced NO release in a biphasic manner, with levels being significantly increased at concentrations over 50 mM. The NO donor, sodium nitroprusside, mimicked the vanadate effect: it inhibited cell growth and alkaline phosphatase activity in a dose-dependent manner. Vanadate enhanced the NO synthases, the endothelial and inducible eNOS and iNOS. isoforms, in a dose-dependent manner. Experiments performed with the ionophore A23187 and EGTA suggested that vanadate-induced NO production involves Ca2q-dependent and -independent mechanisms. Altogether, our results suggest that NO may play a critical role in the bioactivity of vanadium in osteoblast-like cells. q2000 Elsevier Science B.V. All rights reserved. - ArtĆculo
Acceso Abierto Advanced glycation end-products (AGEs) induce concerted changes in the osteoblastic expression of their receptor RAGE and in the activation of extracellular signal-regulated kinases (ERK)(2003) Cortizo, Ana MarĆa; Lettieri, M.G.; Barrio, D.A.; Mercer, N.; Etcheverry, S.B.; McCarthy, Antonio DesmondAn increase in the interaction between advanced glycation end-products (AGEs) and their receptor RAGE is believed to contribute to the pathogenesis of chronic complications of Diabetes mellitus, which can include bone alterations such as osteopenia. We have recently found that extracellular AGEs can directly regulate the growth and development of rat osteosarcoma UMR106 cells, and of mouse calvaria-derived MC3T3E1 osteoblasts throughout their successive developmental stages (proliferation, differentiation and mineralisation), possibly by the recognition of AGEs moieties by specific osteoblastic receptors which are present in both cell lines. In the present study we examined the possible expression of RAGE by UMR106 and MC3T3E1 osteoblastic cells, by immunoblot analysis. We also investigated whether short-, medium- or long-term exposure of osteoblasts to extracellular AGEs, could modify their affinity constant and maximal binding for AGEs (by 125I-AGE-BSA binding experiments), their expression of RAGE (by immunoblot analysis) and the activation status of the osteoblastic ERK 1/2 signal transduction mechanism (by immunoblot analysis for ERK and P-ERK). Our results show that both osteoblastic cell lines express readily detectable levels of RAGE. Short-term exposure of phenotypically mature osteoblastic UMR106 cells to AGEs decrease the cellular density of AGE-binding sites while increasing the affinity of these sites for AGEs. This culture condition also dose-dependently increased the expression of RAGE and the activation of ERK. In proliferating MC3T3E1 pre-osteoblasts, 24-72 h exposure to AGEs did not modify expression of RAGE, ERK activation or the cellular density of AGE-binding sites. However, it did change the affinity of these binding sites forAGEs, with both higher- and lower-affinity sites now being apparent. Medium-term ( 1 week) incubation of differentiated MC3T3E1 osteoblasts with AGEs, induced a simultaneous increase in RAGE expression and in the relative amount of P-ERK. Mineralising MC3T3E1 cultures grown for 3 weeks in the presence of extracellular AGEs showed a decrease both in RAGE and P-ERK expression. These results indicate that, in phenotypically mature osteoblastic cells, changes in ERK activation closely follow the AGEs-induced regulation of RAGE expression. Thus, the AGEs-induced biological effects that we have observed previously in osteoblasts, could be mediated by RAGE in the later stages of development, and mediated by other AGE receptors in the earlier pre-osteoblastic stage. - ArtĆculo
Acceso Abierto Advanced glycation endproducts interfere with integrin-mediated osteoblastic attachment to a type-I collagen matrix(2004) McCarthy, Antonio Desmond; Uemurab, Toshimasa; Etcheverry, Susana B.; Cortizo, Ana MarĆaThe adhesion of osteoblasts to bone extracellular matrix, of which type-I collagen constitutes >85%, can modulate diverse aspects of their physiology such as growth, differentiation and mineralisation. In this study we examined the adhesion of UMR106 rat osteoblast-like cells either to a control (Col) or advanced-glycation-endproduct-modified (AGEs-Col) type I collagen matrix. We investigated the possible role of different integrin receptors in osteoblastic adhesion, by co-incubating these cells either with Ī²-peptide (conserved sequence 113ā125 of the Ī² subunit of integrins) or with two other peptides, RGD (Arg-Gly-Asp) and DGEA (Asp-Gly-Glu-Ala), which are recognition sequences for the Ī±-subunits of Ī±1,5Ī²1and Ī±2Ī²1integrins. Collagen glycation inhibited the adhesion of UMR106 osteoblasts to the matrix (40% reduction versus Col,P<0.001). Ī²-Peptide showed a dose- and glycation-dependent inhibitory effect on adhesion, and at a concentration of 100Ī¼M decreased the attachment of UMR106 cells to both matrices (42% to Col,P<0.001; and 25% to AGEs-Col,P<0.01). The synthetic peptides RGD (1mM) and DGEA (5mM) inhibited the attachment of UMR106 cells to Col (30 and 20%,P<0.01 andP<0.001, respectively), but not to AGEs-Col. Ī²-Peptide induced an increase in UMR106 cell clumping and a decrease in cellular spreading, while DGEA increased spreading with cellular extensions in multiple directions. These results indicate that both Ī± and Ī² integrin subunits participate in osteoblastic attachment to type-I collagen, probably through the Ī±1,5Ī²1and Ī±2Ī²1integrins. AGEs-modification of type-I collagen impairs the integrin-mediated adhesion of osteoblastic cells to the matrix, and could thus contribute to the pathogenesis of diabetic osteopenia. - ArtĆculo
Acceso Abierto AGE-R3/galectin-3 expression in osteoblast-like cells: Regulation by AGEs(2004) Mercer, Natalia; Ahmed, Hafiz; McCarthy, Antonio Desmond; Etcheverry, Susana B.; Vasta, Gerardo R.; Cortizo, Ana MarĆaThe accumulation of irreversible advanced glycation endproducts (AGEs) on long-lived proteins, and the interaction of AGEs with cellular receptors such as AGE-R3/galectin-3 and RAGE, are considered to be key events in the development of longterm complications of diabetes mellitus, Alzheimerās disease, uremia and ageing. The aim of this study was to investigate the expression and sub-cellular distribution of galectin-3, as well as its possible modulation by AGEs, in MC3T3E1 mouse calvaria-derived osteoblasts and in UMR 106 rat osteosarcoma cells. Both osteoblastic lines were cultured either with control bovine serum albumin (BSA) or with AGEs-BSA for 48 h. Cells were evaluated for galectin-3 expression by fixing and immunofluorescent microscopic analysis; or Western blot analysis of whole cell extracts, sub-cellular fractions and culture media. Both cell lines express 30 kDa (monomeric) galectin-3, although expression was about 15-fold lower in the UMR106 osteosarcoma cells. Dimeric (70 kDa) galectin-3 was additionally observed in the UMR106 cells. Immunofluorescent analysis of galectin-3 distribution showed a diffuse cytoplasmic and strong nuclear pattern in MC3T3E1 osteoblasts, and a patchy cytoplasmic pattern in UMR106 cells. Western blot analysis for both cell lines showed that galectin-3 was mainly found in the cytoplasm and in minor amounts in the microsomal fraction, while considerable amounts were secreted into the culture media. Exposure to 100ā200 Ī¼g/mL AGEs-BSA increased the cellular content of 30 kDa galectin-3 (20ā25% for MC3T3E1 and 35ā70% for UMR106 versus control BSA, p < 0.05), and decreased the culture media levels of galectin-3 (10ā20% for MC3T3E1 and for UMR106 versus control BSA, p < 0.05). These results confirm the expression of galectin-3 in osteoblastic cells, and suggest different levels and sub-cellular distribution of this protein in transformed versus non-transformed osteoblasts. Osteoblastic exposure to AGEs alters their expression and secretion of galectin-3, which could have significant consequences on osteoblast metabolism and thus on bone turnover. - ArtĆculo
Acceso Abierto Osteogenic activity of vanadyl(IV)āascorbate complex: evaluation of its mechanism of action(2006) Cortizo, Ana MarĆa; Molinuevo, M. Silvina; Barrio, Daniel A.; Bruzzone, LilianaWe have previously shown that different vanadium(IV) complexes regulate osteoblastic growth. Since vanadium compounds are accumulated in vivo in bone, they may affect bone turnover. The development of vanadium complexes with different ligands could be an alternative strategy of use in skeletal tissue engineering. In this study, we have investigated the osteogenic properties of a vanadyl(IV)āascorbate (VOAsc) complex, as well as its possible mechanisms of action, on two osteoblastic cell lines in culture. VOAsc (2.5ā25 M) significantly stimulated osteoblastic proliferation (113ā125% basal, p < 0.01) in UMR106 cells, but not in the MC3T3E1 cell line. VOAsc (5ā100 M) dose-dependently stimulated type-I collagen production (107ā156% basal) in osteoblasts. After 3 weeks of culture, 5ā25 M VOAsc increased the formation of nodules of mineralization in MC3T3E1 cells (7.7ā20-fold control, p < 0.001). VOAsc (50ā100 M) significantly stimulated apoptosis in both cell lines (170ā230% basal, p < 0.02ā0.002), but did not affect reactive oxygen species production. The complex inhibited alkaline and neutral phosphatases from osteoblastic extracts with semi-maximal effect at 10 M doses. VOAsc induced the activation and redistribution of P-ERK in a time- and dose-dependent manner. Inhibitors of the mitogen activated protein kinases (MAPK) pathway (PD98059 and UO126) partially blocked the VOAsc-enhanced osteoblastic proliferation and collagen production. In addition, wortmanin, a PI-3-K inhibitor and type-L channel blocker nifedipine also partially abrogated these effects of VOAsc on osteoblasts. Our in vitro results suggest that this vanadyl(IV)āascorbate complex could be a useful pharmacological tool for bone tissue regeneration. - ArtĆculo
Acceso Abierto Osteogenic actions of the anti-diabetic drug metformin on osteoblasts in culture(2006) Cortizo, Ana MarĆa; Sedlinsky, Claudia; McCarthy, Antonio Desmond; Blanco, Alcira; Schurman, LeĆ³nAn association has been previously established between uncompensated diabetes mellitus and the loss of bone mineral density and/or quality. In this study, we evaluated the effects of metformin on the growth and differentiation of osteoblasts in culture. Treatment of two osteoblast-like cells (UMR106 and MC3T3E1) with metformin (25ā500Ī¼M) for 24h led to a dose-dependent increase of cell proliferation. Metformin also promoted osteoblastic differentiation: it increased type-I collagen production in both cell lines and stimulated alkaline phosphatase activity in MC3T3E1 osteoblasts. In addition, metformin markedly increased the formation of nodules of mineralization in 3-week MC3T3E1 cultures. Metformin induced activation and redistribution of phosphorylated extracellular signal-regulated kinase (P-ERK) in a transient manner, and dosedependently stimulated the expression of endothelial and inducible nitric oxide synthases (e/iNOS). These results show for the first time a direct osteogenic effect of metformin on osteoblasts in culture, which could be mediated by activation/redistribution of ERK-1/2 and induction of e/ iNOS. - ArtĆculo
Acceso Abierto Involvement of integrins in the adhesion of osteoblastic cells to a type-I collagen matrix(2006) McCarthy, Antonio Desmond; Uemura, Toshimasa; Etcheverry, Susana B.; Cortizo, Ana MarĆaSe han desarrollado varios biomateriales con potencial aplicaci Ć³n en la reconstrucciĆ³n de tejidos. En este sentido, existe un creciente interĆ©s en el diseƱo de materiales de implante Ć³seo con mĆ”xima biocompatibilidad y adecuada adhesividad celular. El objetivo de este trabajo fue estudiar cuĆ”les receptores integrinas participan en la adhesiĆ³n de osteoblastos a una matriz de colĆ”geno tipo-I. Se analizaron dos lĆneas celulares osteoblĆ”sticas: UMR106, derivada de osteosarcoma de rata; y MC3T3E1, derivada de calvaria de rata. Las cĆ©lulas se cultivaron durante una hora sobre plĆ”stico, o sobre un gel de colĆ”geno tipo-I, solas o co-incubadas con diferentes pĆ©ptidos: (a) pĆ©ptido-b, un oligopĆ©ptido de 13 aminoĆ”cidos que corresponde a la secuencia conservada 113-125 de la subunidad b de los receptores integrinas; (b) RGD (Arg-Gly-Asp), que corresponde a la secuencia de reconocimiento de la subunidad a de las integrinas a1,5b1; o (c) DGEA (Asp-Gly-Glu-Ala), la secuencia de reconocimiento de la subunidad a de las integrinas a2b1. La adhesiĆ³n y la inducciĆ³n de extensiones celulares se evaluaron microscĆ³picamente luego de lavar, fijar y colorear las cĆ©lulas con Giemsa. Los resultados demostraron que las cĆ©lulas osteoblĆ”sticas se adhieren mĆ”s fĆ”cilmente a un sustrato de colĆ”geno tipo-I que al plĆ”stico (86 Ā± 5 cĆ©lulas/campo vs. 69 Ā± 4 cĆ©lulas/campo para colĆ”geno tipo-I vs. plĆ”stico, respectivamente, p < 0,02). El pĆ©ptido-b inhibiĆ³ la adhesiĆ³n de ambas lĆneas celulares a una matriz de colĆ”geno y el efecto inhibidor mostrĆ³ dependencia dosis-respuesta. Por otro lado, este pĆ©ptido indujo en las cĆ©lulas tipo osteoblastos UMR106 un aumento del agrupamiento intercelular, y una reducciĆ³n en la inducciĆ³n de extensiones celulares. Estos cambios morfol Ć³gicos podrĆan estar indicando un incremento en las interacciones cĆ©lulac Ć©lula y una disminuciĆ³n en las interacciones cĆ©lula-matriz, posiblemente inducidos por el pĆ©ptido-b. De forma similar, los pĆ©ptidos RGD y DGEA disminuyeron significativamente, entre un 20 y 30 %, la adhesiĆ³n de las cĆ©lulas UMR106 y MC3T3E1 a la matriz de colĆ”geno. Las cĆ©lulas UMR106 cultivadas sobre colĆ”geno en presencia de DGEA mostraron un mayor estiramiento citoplasmĆ”tico, con inducciĆ³n de extensiones celulares en mĆŗltiples direcciones. En suma, estos resultados sugieren que las subunidades a y b de varios receptores integrinas estĆ”n involucradas en la adhesiĆ³n de las cĆ©lulas tipo osteoblastos a una matriz de colĆ”geno tipo-I. AsĆ, el recubrimiento de ciertos biomateriales con pĆ©ptidos de reconocimiento o con mol Ć©culas de colĆ”geno intacto, podrĆa mejorar la osteointegraciĆ³n de implantes para la reparaciĆ³n del tejido Ć³seo.