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- ArtículoEmbargadoMultiple modulators of the glucose-induced net calcium uptake by isolated islets(1984) Borelli, María Inés; Cortizo, Ana María; Gagliardino, Elma Edith P. de; Garcia, María Elisa; Gagliardino, Juan JoséGlucose-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.
- ComunicacionAcceso AbiertoEffect of hypothyroidism on the composition and turnover rate of islet phospholipids(1984) Cortizo, Ana María; 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.
- ArtículoAcceso AbiertoMecanismo de acción de hormonas tiroideas sobre el páncreas endócrino(1987) Cortizo, Ana MaríaEl rol de las hormonas tiroideas en la Diabetes Mellitus y la función endocrina del páncreas fue estudiado por primera vez en 1944 por B. A. Houssay (1 ). Trabajando con perros parcialmente panceatectomizados, sometidos a tratamiento con hormonas tiroideas, observó que adquirían una forma de diabetes irreversible, que persistía aún después de interrumpir el tratamiento tiroideo. Llamó a ésta Diabetes Meta.tiroidea ; su mecanismo parecía ser un agotamiento pancreático consecutivo a una sobreestimulación (2). Sus estudios se extendieron en animales sometidos a tiroidectomía y pancreatectomía subtotal simultánea, observando que no se reducía la severidad de la diabetes, sino que la operación exacerbaba los síntomas, la enfermedad progresaba más rápidamente y finalmente morían en hiperglucemia. A partir de estos experimentos, las hormonas tiroideas han sido consideradas como hormonas diabetogénicas. Numerosos investigadores han observado la intolerancia a la glucosa asociada con una disfunción tiroidea, tanto a nivel clínico como experimental (3-11). Pero el mecanismo por el cual las hormonas tiroideas llevan a cabo estos cambios es hasta hoy desconocido.
- ArtículoAcceso AbiertoVectorial insulin secretion by pancreatic β-cells(1990) Cortizo, Ana María; Espinal, Joseph; Hammonds, PeterMorphological studies of pancreatic β-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.
- ArtículoAcceso AbiertoChanges induced by glucose in the plasma membrane properties of pancreatic islets(1990) Cortizo, Ana María; Paladini, A.; Díaz, G.B.; García, 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.
- ArtículoAcceso AbiertoStimulated release of arachidonate and prostaglandins is vectorial in MDCK epithelial cells(1992) Cortizo, Ana María; 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.
- ArtículoAcceso AbiertoComparative Study of IGFBP Properties in Toad and Rat Sera(1993) Cortizo, Ana María; 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 ± 0.48 ng/ml) similar to those previously reported in amphibians but far below those found in rats. IGFBP levels were estimated at 129 ± 23 and 4249 ± 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 serum—as already reported—three 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 × 1010M-1;R1= 1.2 × 10-11M) and another with low affinity-high capacity (Ka2= 1.9 × 108M-1;R2= 1.9 × 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.
- ArtículoAcceso AbiertoVanadium Compounds(1994) Cortizo, Ana María; 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.
- ArtículoAcceso AbiertoVanadium derivatives act as growth factor--mimetic compounds upon differentiation and proliferation of osteoblast-like UMR106 cells(1995) Cortizo, Ana María; 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.
- ArtículoAcceso AbiertoProliferative and morphological changes induced by vanadium compounds on Swiss 3T3 fibroblasts(1997) Cortizo, Ana María; 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 2.5–10 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
- ArtículoAcceso AbiertoInsulin-like growth factor binding proteins from adult-hamster pancreatic islets: influence of glucose concentration(1997) Massa, L.; Cortizo, Ana María; Gagliardino, Juan JoséThis 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.
- ArtículoAcceso AbiertoRelationship 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 María; Lee, P.D.; Cédola, 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.
- ArtículoAcceso AbiertoFactores de crecimiento insulino símiles: estructura, bioactividad y métodos de ensayo(1998) Cortizo, Ana María; Mccarthy, AntonioEl sistema de los factores de crecimiento insulino-símiles (IGF) se halla involucrado en diferentes aspectos de la regulación celular y tisular, como así también en el desarrollo y el crecimiento corporal. Este sistema depende de la interacción entre ligandos (IGF-I, IGF-II), receptores (Tipo I, Tipo II), proteínas ligadoras o transportadoras (IGFBP-1 a -6), y proteasas específicas para las IGFBPs. La acción de los IGFs se encuentra regulada por diferentes factores y estímulos, tales como la hormona de crecimiento, que actúan a diversos niveles. El desarrollo de nuevos métodos para analizar los diferentes componentes del sistema de los IGFs ha aportado elementos adicionales para la evaluación, diagnóstico y seguimiento de pacientes con alteraciones del crecimiento.
- ArtículoAcceso AbiertoVanadate-induced nitric oxide production: role in osteoblast growth and differentiation(2000) Cortizo, Ana María; 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 2.5–100 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.
- ArtículoAcceso AbiertoAdvanced 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 María; 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.
- ArtículoAcceso AbiertoAGE-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 MaríaThe 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, Alzheimer’s 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 100–200 μg/mL AGEs-BSA increased the cellular content of 30 kDa galectin-3 (20–25% for MC3T3E1 and 35–70% for UMR106 versus control BSA, p < 0.05), and decreased the culture media levels of galectin-3 (10–20% 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.
- ArtículoAcceso AbiertoAdvanced 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 MaríaThe 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 β-peptide (conserved sequence 113–125 of the β subunit of integrins) or with two other peptides, RGD (Arg-Gly-Asp) and DGEA (Asp-Gly-Glu-Ala), which are recognition sequences for the α-subunits of α1,5β1and α2β1integrins. Collagen glycation inhibited the adhesion of UMR106 osteoblasts to the matrix (40% reduction versus Col,P<0.001). β-Peptide showed a dose- and glycation-dependent inhibitory effect on adhesion, and at a concentration of 100μM 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. β-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 α and β integrin subunits participate in osteoblastic attachment to type-I collagen, probably through the α1,5β1and α2β1integrins. 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.
- ArtículoAcceso AbiertoInvolvement 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 MaríaSe han desarrollado varios biomateriales con potencial aplicaci ón en la reconstrucción de tejidos. En este sentido, existe un creciente interés en el diseño de materiales de implante óseo con máxima biocompatibilidad y adecuada adhesividad celular. El objetivo de este trabajo fue estudiar cuáles receptores integrinas participan en la adhesión de osteoblastos a una matriz de colágeno tipo-I. Se analizaron dos líneas celulares osteoblásticas: UMR106, derivada de osteosarcoma de rata; y MC3T3E1, derivada de calvaria de rata. Las células se cultivaron durante una hora sobre plástico, o sobre un gel de colágeno tipo-I, solas o co-incubadas con diferentes péptidos: (a) péptido-b, un oligopéptido de 13 aminoácidos 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 adhesión y la inducción de extensiones celulares se evaluaron microscópicamente luego de lavar, fijar y colorear las células con Giemsa. Los resultados demostraron que las células osteoblásticas se adhieren más fácilmente a un sustrato de colágeno tipo-I que al plástico (86 ± 5 células/campo vs. 69 ± 4 células/campo para colágeno tipo-I vs. plástico, respectivamente, p < 0,02). El péptido-b inhibió la adhesión de ambas líneas celulares a una matriz de colágeno y el efecto inhibidor mostró dependencia dosis-respuesta. Por otro lado, este péptido indujo en las células tipo osteoblastos UMR106 un aumento del agrupamiento intercelular, y una reducción en la inducción de extensiones celulares. Estos cambios morfol ógicos podrían estar indicando un incremento en las interacciones célulac élula y una disminución en las interacciones célula-matriz, posiblemente inducidos por el péptido-b. De forma similar, los péptidos RGD y DGEA disminuyeron significativamente, entre un 20 y 30 %, la adhesión de las células UMR106 y MC3T3E1 a la matriz de colágeno. Las células UMR106 cultivadas sobre colágeno en presencia de DGEA mostraron un mayor estiramiento citoplasmático, con inducción de extensiones celulares en múltiples direcciones. En suma, estos resultados sugieren que las subunidades a y b de varios receptores integrinas están involucradas en la adhesión de las células tipo osteoblastos a una matriz de colágeno tipo-I. Así, el recubrimiento de ciertos biomateriales con péptidos de reconocimiento o con mol éculas de colágeno intacto, podría mejorar la osteointegración de implantes para la reparación del tejido óseo.
- ArtículoAcceso AbiertoOsteogenic activity of vanadyl(IV)–ascorbate complex: evaluation of its mechanism of action(2006) Cortizo, Ana María; 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)–ascorbate (VOAsc) complex, as well as its possible mechanisms of action, on two osteoblastic cell lines in culture. VOAsc (2.5–25 M) significantly stimulated osteoblastic proliferation (113–125% basal, p < 0.01) in UMR106 cells, but not in the MC3T3E1 cell line. VOAsc (5–100 M) dose-dependently stimulated type-I collagen production (107–156% basal) in osteoblasts. After 3 weeks of culture, 5–25 M VOAsc increased the formation of nodules of mineralization in MC3T3E1 cells (7.7–20-fold control, p < 0.001). VOAsc (50–100 M) significantly stimulated apoptosis in both cell lines (170–230% basal, p < 0.02–0.002), 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)–ascorbate complex could be a useful pharmacological tool for bone tissue regeneration.
- ArtículoAcceso AbiertoOsteogenic actions of the anti-diabetic drug metformin on osteoblasts in culture(2006) Cortizo, Ana María; Sedlinsky, Claudia; McCarthy, Antonio Desmond; Blanco, Alcira; Schurman, LeónAn 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 (25–500μM) 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.
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