RESUMEN
BACKGROUND: Bone marrow (BM)-derived mesenchymal stromal cells (MSC) are promising candidate cells for the development of neuroregenerative therapies. We have previously introduced the pro-neural conversion of human MSC to neural stem cell-like cells (m-NSC) by culturing them in suspension culture under serum-free conditions. METHODS: In the present study, we used a modified Boyden chamber assay to study the influence of various chemoattractants and extracellular matrix components on MSC and m-NSC migration in vitro. The underlying mechanisms were investigated further by applying real-time reverse transcriptase (RT)-polymerase chain reaction (PCR) and flow cytometry. RESULTS: The basal migration of m-NSC was significantly reduced compared with MSC (six versus 27 out of 10,000 cells migrated within 6 h). We evaluated the effects of bone morphogenic protein 2 (BMP2), insulin-like growth factor 1 (IGF1), platelet-derived growth factor bb (PDGFbb), vascular endothelial growth factor (VEGFa), and stromal cell-derived factor 1 (SDF1) on the migration potential of both cell types and PDGFbb proved to be the most potent stimulant of migration (235 versus 198 m-NSC or MSC migrated). Adhesion of m-NSC to the filter membrane was delayed and not affected by IGF1 or PDGFbb: 90% of MSC, but only 20% of m-NSC, adhered within 1 h, with 90% of m-NSC adhering within 3 h. However, real-time RT-PCR and flow cytometry revealed an up-regulation of the PDGF receptor B following conversion. Coating the membranes with collagen type I or hyaluronan also significantly influenced cell migration. DISCUSSION: We could identify major chemoattractive factors for m-NSC and gained partial insight into the complex processes involved in migration of neurally converted cells.
Asunto(s)
Células Madre Mesenquimatosas/citología , Sistema Nervioso/citología , Proteínas Proto-Oncogénicas c-sis/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/metabolismo , Adolescente , Adulto , Proteína Morfogenética Ósea 2/metabolismo , Adhesión Celular/genética , Técnicas de Cultivo de Célula , Movimiento Celular/genética , Transdiferenciación Celular/genética , Quimiocina CXCL12/metabolismo , Colágeno Tipo I/metabolismo , Femenino , Regulación del Desarrollo de la Expresión Génica , Humanos , Ácido Hialurónico/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Persona de Mediana Edad , Sistema Nervioso/crecimiento & desarrollo , Sistema Nervioso/metabolismo , Receptor beta de Factor de Crecimiento Derivado de Plaquetas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Células del Estroma/citología , Células del Estroma/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
Stem and progenitor cells provide a promising therapeutic strategy for amyotrophic lateral sclerosis (ALS). To comparatively evaluate the therapeutic potentials of human bone marrow-derived mesodermal stromal cells (hMSCs) and umbilical cord blood cells (hUBCs) in ALS, we transplanted hMSCs and hUBCs and their neuroectodermal derivatives (hMSC-NSCs and hUBC-NSCs) into the ALS mouse model over-expressing the G93A mutant of the human SOD1 gene. We used a standardized protocol similar to clinical studies by performing a power calculation to estimate sample size prior to transplantation, matching the treatment groups for gender and hSOD-G93A gene content, and applying a novel method for directly injecting 100,000 cells into the CSF (the cisterna magna). Ten days after transplantation we found many cells within the subarachnoidal space ranging from frontal basal cisterns back to the cisterna magna, but only a few cells around the spinal cord. hMSCs and hMSC-NSCs were also located within the Purkinje cell layer. Intrathecal cell application did not affect survival times of mice compared to controls. Consistently, time of disease onset and first pareses, death weight, and motor neuron count in lumbar spinal cord did not vary between treatment groups. Interestingly, transplantation of hMSCs led to an increase of pre-symptomatic motor performance compared to controls in female animals. The negative outcome of the present study is most likely due to insufficient cell numbers within the affected brain regions (mainly the spinal cord). Further experiments defining the optimal cell dose, time point and route of application and particularly strategies to improve the homing of transplanted cells towards the CNS region of interest are warranted to define the therapeutic potential of mesodermal stem cells for the treatment of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/terapia , Movimiento Celular/fisiología , Columna Vertebral/fisiología , Trasplante de Células Madre , Envejecimiento/fisiología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/fisiopatología , Animales , Recuento de Células , Supervivencia Celular , Cisterna Magna/fisiología , Progresión de la Enfermedad , Humanos , Ratones , Ratones Transgénicos , Desempeño Psicomotor/fisiología , Tamaño de la Muestra , Espacio Subaracnoideo/fisiología , Superóxido Dismutasa/genéticaRESUMEN
Cu/Zn SOD1(G93A) transgenic mice develop phenotypical hallmarks of ALS and serve therefore as an established model to study the molecular mechanisms underlying this disease. Recent reports demonstrate that mutations in the motor protein dynein in Legs at odd angles (Loa) and Cramping (Cra1) mice lead to similar but milder phenotypes. Surprisingly, double transgenic mice (Loa/SOD1(G93A)) have been recently shown to attenuate rather than to accelerate the phenotypical expression of motor neuron degeneration. These results raise the question whether other functional relevant mutations in dynein cause a similar effect. To address this question, we have cross-bred SOD1(G93A) with Cra1/+ mice. These double transgenic mice show an attenuated decline of both motor activity and body weight and an increase of survival time compared to SOD1(G93A) mice. Thus, this study confirms that mechanisms associated with dynein such as retrograde axonal transport may play an important role in SOD1(G93A-) toxicity on motor neurons.
Asunto(s)
Dineínas/genética , Regulación de la Expresión Génica/genética , Enfermedad de la Neurona Motora/genética , Mutación , Degeneración Nerviosa/fisiopatología , Animales , Peso Corporal/genética , Recuento de Células/métodos , Modelos Animales de Enfermedad , Dineínas/clasificación , Masculino , Ratones , Ratones Transgénicos , Actividad Motora/genética , Enfermedad de la Neurona Motora/fisiopatología , Tiempo de Reacción/genética , Superóxido Dismutasa/genéticaRESUMEN
Tetrahydrobiopterin [(6R)-5,6,7,8-tetrahydro-L-biopterin, H(4)biopterin] is one of several cofactors of nitric oxide synthases (EC 1.14.13.39). Here we compared the action of N(5)-substituted derivatives on recombinant rat neuronal nitric oxide synthase with their effects on dihydropteridine reductase (EC 1.6.99.7) and phenylalanine hydroxylase (EC 1.14.16.1),the well-studied classical H(4)biopterin-dependent reactions. H(4)biopterin substituted at N(5) with methyl, hydroxymethyl, formyl and acetyl groups were used. Substitution at N(5) occurs at a position critical to the redox cycle of the cofactor in phenylalanine hydroxylase/dihydropteridine reductase. We also included N(2)'-methyl H(4)biopterin, a derivative substituted at a position not directly involved in redox cycling, as a control. As compared with N(5)-methyl H(4)biopterin, N(5)-formyl H(4)biopterin bound with twice the capacity but stimulated nitric oxide synthase to a lesser extent. Depending on the substituent used, N(5)-substituted derivatives were redox-active: N(5)-methyl- and N(5)-hydroxyl methyl H(4)biopterin, but not N(5)-formyl- and N(5)-acetyl H(4)biopterin, reduced 2,6-dichlorophenol indophenol. N(5)-Substituted H(4)biopterin derivatives were not oxidized to products serving as substrates for dihydropteridine reductase and,depending on the substituent, were competitive inhibitors of phenylalanine hydroxylase: N(5)-methyl- and N(5)-hydroxymethyl H(4)biopterin inhibited phenylalanine hydroxylase, whereas N(5)-formyl- and N(5)-acetyl H(4)biopterin had no effect. Our data demonstrate differences in the mechanism of stimulation of phenylalanine hydroxylase and nitric oxide synthase by H(4)biopterin. They are compatible with a novel, non-classical, redox-active contribution of H(4)biopterin to the catalysis of the nitric oxide synthase reaction.
Asunto(s)
Biopterinas/análogos & derivados , Dihidropteridina Reductasa/efectos de los fármacos , Óxido Nítrico Sintasa/efectos de los fármacos , Fenilalanina Hidroxilasa/efectos de los fármacos , Animales , Biopterinas/química , Biopterinas/farmacología , Dihidropteridina Reductasa/metabolismo , Activación Enzimática , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo I , Fenilalanina Hidroxilasa/metabolismo , Ratas , Proteínas Recombinantes/efectos de los fármacos , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Endothelial nitric-oxide synthase (type III) (eNOS) was reported to form an inhibitory complex with the bradykinin receptor B2 (B2R) from which the enzyme is released in an active form upon receptor activation (Ju, H., Venema, V. J., Marrero, M. B., and Venema, R. C. (1998) J. Biol. Chem. 273, 24025-24029). Using a synthetic peptide derived from the known inhibitory sequence of the B2R (residues 310-329) we studied the interaction of the receptor with purified eNOS and neuronal nitric-oxide synthase (type I) (nNOS). The peptide inhibited formation of L-citrulline by eNOS and nNOS with IC(50) values of 10.6 +/- 0.4 microM and 7.1 +/- 0.6 microM, respectively. Inhibition was not due to an interference of the peptide with L-arginine or tetrahydrobiopterin binding. The NADPH oxidase activity of nNOS measured in the absence of L-arginine was inhibited by the peptide with an IC(50) of 3.7 +/- 0.6 microM, but the cytochrome c reductase activity of the enzyme was much less susceptible to inhibition (IC(50) >0.1 mM). Steady-state absorbance spectra of nNOS recorded during uncoupled NADPH oxidation showed that the heme remained oxidized in the presence of the synthetic peptide consisting of amino acids 310-329 of the B2R, whereas the reduced oxyferrous heme complex was accumulated in its absence. These data suggest that binding of the B2R 310-329 peptide blocks flavin to heme electron transfer. Co-immunoprecipitation of B2R and nNOS from human embryonic kidney cells stably transfected with human nNOS suggests that the B2R may functionally interact with nNOS in vivo. This interaction of nNOS with the B2R may recruit the enzyme to allow for the effective coupling of bradykinin signaling to the nitric oxide pathway.
Asunto(s)
Endotelio Vascular/enzimología , Neuronas/enzimología , Óxido Nítrico Sintasa/metabolismo , Receptores de Bradiquinina/metabolismo , Animales , Sitios de Unión , Western Blotting , Línea Celular , Citrulina/metabolismo , Relación Dosis-Respuesta a Droga , Humanos , Cinética , Óxido Nítrico Sintasa/antagonistas & inhibidores , Péptidos/metabolismo , Pruebas de Precipitina , Unión Proteica , Ratas , Receptor de Bradiquinina B2 , Proteínas Recombinantes/metabolismo , Factores de TiempoRESUMEN
Tetrahydrobiopterin ((6R)-5,6,7,8-tetrahydro-L-biopterin (H4biopterin)) is an essential cofactor of nitric-oxide synthases (NOSs), but its role in enzyme function is not known. Binding of the pterin affects the electronic structure of the prosthetic heme group in the oxygenase domain and results in a pronounced stabilization of the active homodimeric structure of the protein. However, these allosteric effects are also produced by the potent pterin antagonist of NOS, 4-amino-H4biopterin, suggesting that the natural cofactor has an additional, as yet unknown catalytic function. Here we show that the 5-methyl analog of H4biopterin, which does not react with O2, is a functionally active pterin cofactor of neuronal NOS. Activation of the H4biopterin-free enzyme occurred in a biphasic manner with half-maximally effective concentrations of approximately 0.2 microM and 10 mM 5-methyl-H4biopterin. Thus, the affinity of the 5-methyl compound was 3 orders of magnitude lower than that of the natural cofactor, allowing the direct demonstration of the functional anticooperativity of the two pterin binding sites of dimeric NOS. In contrast to H4biopterin, which inactivates nitric oxide (NO) through nonenzymatic superoxide formation, up to 1 mM of the 5-methyl derivative did not consume O2 and had no effect on NO steady-state concentrations measured electrochemically with a Clark-type NO electrode. Therefore, reconstitution with 5-methyl-H4biopterin allowed, for the first time, the detection of enzymatic NO formation in the absence of superoxide or NO scavengers. These results unequivocally identify free NO as a NOS product and indicate that reductive O2 activation by the pterin cofactor is not essential to NO biosynthesis.