Isolation of a small fraction of dermatan sulfate with potencial therapeutical application

El descubrimiento del cofactor II de la heparina (HCoII), desplazó el enfoque de las investigaciones sobre el mecanismo de la coagulación desde la heparina, que se encuentra fundamentalmente en los mastocitos, hacia el dermatán sulfato (DS) que posee una distribución mucho más amplia en los tejidos y en las paredes de los vasos sanguíneos. El nuevo cofactor de la heparina es el único cuya actividad anticoagulante es estimulada por el DS, en concentraciones diez veces menores que la heparina y el heparán sulfato (HS). Se ha descripto el aislamiento de fracciones de la heparina con muy alta actividad anticoagulante a través de la interacción con la Concanavalina A y con la primera proteína del sistema del complemento (C1). En ambos casos fueron imprescindibles condiciones de baja fuerza iónica y presencia de iones calcio. Los experimentos con la heparina se extendieron al DS en presencia del C1, utilizando las mismas condiciones experimentales descriptas, y se determinaron la actividad biológica y el grado de sulfatación de las subpoblaciones aisladas después de la interacción. Se obtuvo en el precipitado una fracción del DS de bajo peso molecular con una actividad anticoagulante cuatro veces mayor que el material inicial, un contenido en sulfatos aproximadamente tres veces mayor, y tres veces más capacidad de inhibición de la formación de trombos. El peso molecular de esta fracción es inferior a 8.000 Daltons.Heparin is mainly found in mastocytes, and during many decades the studies of the coagulation mechanism were focused on this hetero-polysaccaride. Dermatan sulfate has a more abundant distribution on tissues and on the blood vascular system. On the other hand, the anticoagulant activity of the second cofactor of heparin is stimulated by dermatan sulfate, but using ten times less concentration than heparin. Our earlier experiments with heparin and Concanavalin A, and heparin and the first protein of the complement system, C1, have shown the isolation in the heparin molecule by precipitation with both proteins of fractions with very high anticoagulant activity. In both cases the use of very low ionic strength and the presence of calcium ions were necesary. We have extended those experiments to the interaction with dermatan sulfate, and we have also obtained a fraction of dermatan sulfate in the precipitate with very low molecular weight, and with four times higher anticoagulant activity than the fraction that remains in the supernatant. The sulfate content of this fraction is three times higher, as well as its capacity for thrombous inhibition. Molecular weight of this fraction is 8000 Daltons.Fil: Recondo, Eduardo Francisco. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; ArgentinaFil: Calabrese, Graciela Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; ArgentinaFil: Alberto, Maria Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Lazzari, María Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Academia Nacional de Medicina de Buenos Aires; ArgentinaFil: Fernandez, Marta Elena del Valle. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas. Cátedra de Biología Celular y Molecular; Argentin

Learning from Synthetic Models of Extracellular Matrix; Differential Binding of Wild Type and Amyloidogenic Human Apolipoprotein A-I to Hydrogels Formed from Molecules Having Charges Similar to Those Found in Natural GAGs

Among other components of the extracellular matrix (ECM), glycoproteins and glycosaminoglycans (GAGs) have been strongly associated to the retention or misfolding of different proteins inducing the formation of deposits in amyloid diseases. The composition of these molecules is highly diverse and a key issue seems to be the equilibrium between physiological and pathological events. In order to have a model in which the composition of the matrix could be finely controlled, we designed and synthesized crosslinked hydrophilic polymers, the so-called hydrogels varying the amounts of negative charges and hydroxyl groups that are prevalent in GAGs. We checked and compared by fluorescence techniques the binding of human apolipoprotein A-I and a natural mutant involved in amyloidosis to the hydrogel scaffolds. Our results indicate that both proteins are highly retained as long as the negative charge increases, and in addition it was shown that the mutant is more retained than the Wt, indicating that the retention of specific proteins in the ECM could be part of the pathogenicity. These results show the importance of the use of these polymers as a model to get deep insight into the studies of proteins within macromolecules.Fil: Rosu, Silvana Antonia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; ArgentinaFil: Toledo, Leandro. Universidad de Concepción; ChileFil: Urbano, Bruno F.. Universidad de Concepción; ChileFil: Sanchez Donoso, Susana Angelica. Universidad de Concepción; ChileFil: Calabrese, Graciela Cristina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica; ArgentinaFil: Tricerri, Maria Alejandra. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Bioquímicas de La Plata "Prof. Dr. Rodolfo R. Brenner". Universidad Nacional de la Plata. Facultad de Ciencias Médicas. Instituto de Investigaciones Bioquímicas de La Plata ; Argentin

VLDL induced modulation of nitric oxide signalling and cell redox homeostasis in huvec

High levels of circulating lipoprotein constitute a risk factor for cardiovascular diseases, and in this context, the specific role of the very-low-density lipoproteins (VLDL) is poorly understood. The response of human umbilical vein endothelial cells (HUVEC) to VLDL exposure was studied, especially focusing on the pathways involved in alteration of redox homeostasis and nitric oxide (NO) bioavailability. The results obtained by the analysis of the expression level of genes implicated in the NO metabolism and oxidative stress response indicated a strong activation of inducible NO synthase (iNOS) upon 24 h exposure to VLDL, particularly if these have been preventively oxidised. Simultaneously, both mRNA and protein expression of endothelial NO synthase (eNOS) were decreased and its phosphorylation pattern, at the key residues Tyr495 and Ser1177, strongly suggested the occurrence of the eNOS uncoupling. The results are consistent with the observed increased production of nitrites and nitrates (NOx), reactive oxygen species (ROS), 3-nitrotyrosine (3-NT), and, at mitochondrial level, a deficit in mitochondrial O2 consumption. Altogether, these data suggest that the VLDL, particularly if oxidised, when allowed to persist in contact with endothelial cells, strongly alter NO bioavailability, affecting redox homeostasis and mitochondrial function

A small fraction of dermatan sulfate with significantly increased anticoagulant activity was selected by interaction with the first complement protein

Dermatan sulfate (DS) is a member of the family of structurally complex, sulfated, linear heteropolysaccharides called glycosaminoglycans (GAGs). It has a similar structure to heparin and heparan sulfate (HS), but with acetylgalactosamine replacing glucosamine, and the uronic acid moiety, mainly iduronic, joined 1→3 to the hexosamine. We are studying the relationships between structure and activities of dermatan sulfate, in particular those associated with the thrombin inhibition mediated by heparin cofactor II (HCII). As we have demonstrated with heparin, a small fraction of dermatan sulfate was isolated by precipitation with the first component of the complement system, under very specific conditions of low ionic strength, and the presence of calcium ions. The sulfate content and the anticoagulant activity of the dermatan sulfate fraction isolated in the precipitate were three and four times greater respectively than the starting material. Our in vivo studies showed that this fraction has threefold higher thrombolytic activity than the DS. All these results suggest that this fraction could be used as a therapeutic agent for thrombi dissolution. © 2004 Elsevier Ltd. All rights reserved.Fil: Calabrese, Graciela Cristina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; ArgentinaFil: Alberto, Maria Fabiana. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Tubio, Maria Rosario. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; ArgentinaFil: Marani, Mariela Mirta. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; ArgentinaFil: Fernández De Recondo, Marta E.. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; ArgentinaFil: Lazzari, María Ángela. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Medicina Experimental. Academia Nacional de Medicina de Buenos Aires. Instituto de Medicina Experimental; ArgentinaFil: Recondo, Eduardo F.. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales; Argentin

Targeted Anti-Inflammatory Peptide Delivery in Injured Endothelial Cells Using Dermatan Sulfate/Chitosan Nanomaterials

This work describes a novel delivery system for targeting egg-derived anti-inflammatory tripeptide Ile-Arg-Trp (IRW) to endothelial cells. The nanomedicine is synthesized by a simple and reproducible ionotropic gelification method that results in the efficient loading of the positively charged IRW within the dermatan sulfate/ chitosan matrix, as demonstrated by ss-NMR spectroscopy. The incorporation of IRW results in a stable nanoparticle dispersion with a single size population of 442 ± 43 nm. Fluorescence microscopy studies demonstrate the capacity of the nanomaterial to distinguish between a quiescent and an injured endothelium through the interaction of dermatan sulfate with the CD44 receptor. Remarkably, no additional surface functionalization is required as dermatan sulfate mediates their internalization and the intracellular release of this natural anti-inflammatory tripeptide to modulate endothelial inflammatory response. This simple, scalable, and versatile nanotechnology platform opens new opportunities to apply in the therapy of vascular disease.Fil: Blachman, Agustín. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; ArgentinaFil: Funez, Florencia. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; ArgentinaFil: Birocco, Ariadna María. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; ArgentinaFil: Saavedra, Soledad Lorena. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Lazaro Martinez, Juan Manuel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Metabolismo del Fármaco. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Metabolismo del Fármaco; ArgentinaFil: Camperi, Silvia Andrea. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Glisoni, Romina Julieta. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Sosnik, Alejandro Dario. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Nanobiotecnología. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Nanobiotecnología; ArgentinaFil: Calabrese, Graciela Cristina. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Ciencias Biológicas; Argentin