RESUMEN
Glaucoma is a progressive ocular syndrome characterized by degeneration of the optic nerve and irreversible visual field loss. Elevated intraocular pressure (IOP) is the main risk factor for glaucoma. Increased IOP is the result of an imbalance between synthesis and outflow of aqueous humor (AH). Blocking ß2 adrenergic receptor (ADRB2) has shown to reduce IOP by decreasing production of AH at the ciliary body (CB). SYL040012 is a siRNA designed to specifically silence ADRB2 currently under development for glaucoma treatment. Here, we show that SYL040012 specifically reduces ADRB2 expression in cell cultures and eye tissues. The compound enters the eye shortly after administration in eye drops and is rapidly distributed among structures of the anterior segment of the eye. In addition, SYL040012 is actively taken up by cells of the CB but not by cells of systemic organs such as the lungs, where inhibition of ADRB2 could cause undesirable side effects. Moreover, SYL040012 reduces IOP in normotensive and hypertensive animal models and the effect appears to be long lasting and extremely well tolerated both locally and systemically.
Asunto(s)
Glaucoma/genética , Glaucoma/terapia , ARN Interferente Pequeño/genética , Receptores Adrenérgicos beta 2/genética , Animales , Línea Celular , Supervivencia Celular/genética , Ojo/metabolismo , Femenino , Silenciador del Gen , Humanos , Presión Intraocular/genética , Macaca fascicularis , Masculino , Estabilidad del ARN , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/química , ARN Interferente Pequeño/metabolismo , Conejos , Receptores Adrenérgicos beta 2/metabolismo , Distribución TisularRESUMEN
RNA interference is an endogenous mechanism present in most eukaryotic cells that enables degradation of specific mRNAs. Pharmacological exploitation of this mechanism for therapeutic purposes attracted a whole amount of attention in its initial years, but was later hampered due to difficulties in delivery of the pharmacological agents to the appropriate organ or tissue. Advances in recent years have to a certain level started to address this specific issue. Genetic diseases are caused by aberrations in gene sequences or structure; these particular abnormalities are in theory easily addressable by RNAi therapeutics. Sequencing of the human genome has largely contributed to the identification of alterations responsible for genetic conditions, thus facilitating the design of compounds that can address these diseases. This review addresses the currently on-going programs with the aim of developing RNAi and other antisense compounds for the treatment of genetic conditions and the pros and cons that these products may encounter along the way. The authors have focused on those programs that have reached clinical trials or are very close to do so.
Asunto(s)
Elementos sin Sentido (Genética)/uso terapéutico , Enfermedades Genéticas Congénitas/terapia , Oligonucleótidos/uso terapéutico , Interferencia de ARN , ARN Interferente Pequeño/uso terapéutico , Humanos , ARN Interferente Pequeño/genéticaRESUMEN
The GTPase RhoA is a downstream target of heterotrimeric G(13) proteins and plays key roles in cell migration and invasion. Here, we show that expression in human melanoma cells of a constitutively active, GTPase-deficient Galpha(13) form (G(alpha)(13)QL) or lysophosphatidylcholine (LPC)-promoted signaling through G(alpha)(13)-coupled receptors led to a blockade of chemokine-stimulated RhoA activation and cell invasion that was rescued by active RhoA. Melanoma cells expressing G(alpha)(13)QL or cells stimulated with LPC displayed an increase in p190RhoGAP activation, and defects in RhoA activation and invasion were recovered by knocking down p190RhoGAP expression, thus identifying this GTPase-activating protein (GAP) protein as a downstream G(alpha)(13) target that is responsible for these inhibitory responses. In addition, defective stress fiber assembly and reduced migration speed underlay inefficient invasion of G(alpha)(13)QL melanoma cells. Importantly, G(alpha)(13)QL expression in melanoma cells led to impairment in lung metastasis associated with prolonged survival in SCID mice. The data indicate that G(alpha)(13)-dependent downstream effects on RhoA activation and invasion tightly depend on cell type-specific GAP activities and that G(alpha)(13)-p190RhoGAP signaling might represent a potential target for intervention in melanoma metastasis.
Asunto(s)
Subunidades alfa de la Proteína de Unión al GTP G12-G13/fisiología , Factores de Intercambio de Guanina Nucleótido/fisiología , Melanoma/patología , Proteínas Represoras/fisiología , Proteína de Unión al GTP rhoA/antagonistas & inhibidores , Animales , Línea Celular Tumoral , Quimiocina CXCL12/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/fisiología , Humanos , Neoplasias Pulmonares/prevención & control , Neoplasias Pulmonares/secundario , Lisofosfatidilcolinas/farmacología , Melanoma/secundario , Ratones , Ratones SCID , Invasividad Neoplásica , Proteínas Proto-Oncogénicas c-vav/metabolismo , Transducción de Señal , Células 3T3 Swiss , Tromboxano A2/fisiología , Proteína de Unión al GTP rhoA/fisiologíaRESUMEN
The chemokine CXCL12 promotes T lymphocyte adhesion mediated by the integrin alpha4beta1. CXCL12 activates the GTPase Rac, as well as Vav1, a guanine-nucleotide exchange factor for Rac, concomitant with up-regulation of alpha4beta1-dependent adhesion. Inhibition of CXCL12-promoted Rac and Vav1 activation by transfection of dominant negative Rac or Vav1 forms, or by transfection of their siRNA, remarkably impaired the increase in T lymphocyte attachment to alpha4beta1 ligands in response to this chemokine. Importantly, inhibition of Vav1 expression by RNA interference resulted in a blockade of Rac activation in response to CXCL12. Adhesions in flow chambers and soluble binding assays using these transfectants indicated that initial ligand binding and adhesion strengthening mediated by alpha4beta1 were dependent on Vav1 and Rac activation by CXCL12. Finally, CXCL12-promoted T-cell transendothelial migration involving alpha4beta1-mediated adhesion was notably inhibited by expression of dominant negative Vav1 and Rac. These results indicate that activation of Vav1-Rac signaling pathway by CXCL12 represents an important inside-out event controlling efficient up-regulation of alpha4beta1-dependent T lymphocyte adhesion.
Asunto(s)
Integrina alfa4beta1/metabolismo , Proteínas Proto-Oncogénicas c-vav/metabolismo , Linfocitos T/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Western Blotting , Adhesión Celular , Línea Celular , Movimiento Celular , Quimiocina CXCL12 , Quimiocinas/metabolismo , Quimiocinas CXC/metabolismo , Electroforesis en Gel de Poliacrilamida , Endotelio Vascular/citología , GTP Fosfohidrolasas/metabolismo , Genes Dominantes , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Inmunoprecipitación , Ligandos , Linfocitos/citología , Microscopía Confocal , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunología , Factores de Tiempo , Transfección , Regulación hacia ArribaRESUMEN
The chemokine stromal cell-derived factor-1alpha (SDF-1alpha) is expressed by bone marrow (BM) stromal cells and plays key roles in cell homing to and retention into the bone marrow. In multiple myeloma, blood-borne malignant plasma cells home to the BM and accumulate in contact with stromal cells, implicating myeloma cell migration across endothelium. Myeloma cells express the SDF-1alpha receptor CXCR4, as well as the integrin alpha4beta1, which mediates their attachment to BM stroma. We show here that SDF-1alpha promotes transendothelial migration of purified BM myeloma cells and myeloma-derived NCI-H929 cells, involving a transient upregulation of alpha4beta1-dependent cell adhesion to the endothelium. Characterization of intracellular signaling pathways involved in the modulation by SDF-1alpha of alpha4beta1-mediated myeloma cell adhesion revealed that intracellular cAMP amounts associated with the activation of protein kinase A play key roles in this modulation. Furthermore, a functional link between cAMP actions on the dynamics of actin cytoskeleton, RhoA activation, and alpha4beta1-dependent cell adhesion in response to SDF-1alpha has been found. The regulation of alpha4beta1-mediated myeloma cell adhesion by SDF-1alpha could play key roles during myeloma cell homing into and trafficking inside the BM, and characterization of the molecular events involved in SDF-1alpha-activated modulation of this adhesion will contribute to a better understanding of mechanisms participating in cell migration.
Asunto(s)
Células de la Médula Ósea/metabolismo , Movimiento Celular/genética , Quimiocinas CXC/metabolismo , Integrina alfa4beta1/metabolismo , Mieloma Múltiple/metabolismo , Células del Estroma/metabolismo , Citoesqueleto de Actina/metabolismo , Adhesión Celular/genética , Línea Celular Tumoral , Quimiocina CXCL12 , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células Endoteliales/metabolismo , Humanos , Mieloma Múltiple/genética , Mieloma Múltiple/fisiopatología , Receptores CXCR4/metabolismo , Regulación hacia Arriba/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Chemokine stromal cell-derived factor-1 (SDF-1) is expressed by bone marrow (BM) stromal cells and plays key roles in BM cell migration. Modulation of its expression could affect the migratory capacity of cells trafficking the BM, such as hematopoietic progenitor and leukemic cells. Transforming growth factor-beta1 (TGF-beta1) is present in the BM environment and constitutes a pivotal molecule controlling BM cell proliferation and differentiation. We used the BM stromal cell line MS-5 as a model to investigate whether SDF-1 expression constitutes a target for TGF-beta1 regulation and its functional consequences. We show here that TGF-beta1 down-regulates SDF-1 expression, both at the mRNA level, involving a decrease in transcriptional efficiency, and at the protein level, as detected in lysates and supernatants from MS-5 cells. Reduction of SDF-1 in supernatants from TGF-beta1-treated MS-5 cells correlated with decreased, SDF-1-dependent, chemotactic, and transendothelial migratory responses of the BM model cell lines NCI-H929 and Mo7e compared with their responses to supernatants from untreated MS-5 cells. In addition, supernatants from TGF-beta1-exposed MS-5 cells had substantially lower efficiency in promoting integrin alpha4beta1-mediated adhesion of NCI-H929 and Mo7e cells to soluble vascular cell adhesion molecule-1 (sVCAM-1) and CS-1/fibronectin than their untreated counterparts. Moreover, human cord blood CD34+ hematopoietic progenitor cells displayed SDF-1-dependent reduced responses in chemotaxis, transendothelial migration, and up-regulation of adhesion to sVCAM-1 when supernatants from TGF-beta1-treated MS-5 cells were used compared with supernatants from untreated cells. These data indicate that TGF-beta1-controlled reduction in SDF-1 expression influences BM cell migration and adhesion, which could affect the motility of cells trafficking the bone marrow.
Asunto(s)
Adhesión Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Quimiocinas CXC/genética , Células del Estroma/citología , Factor de Crecimiento Transformador beta/farmacología , Células 3T3 , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/efectos de los fármacos , Quimiocina CXCL12 , Quimiocinas CXC/metabolismo , Regulación hacia Abajo/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Humanos , Leucemia Megacarioblástica Aguda , Ratones , Mieloma Múltiple , ARN Mensajero/análisis , Células del Estroma/efectos de los fármacos , Factor de Crecimiento Transformador beta1 , Células Tumorales CultivadasRESUMEN
The interaction between the integrin alpha(4)beta(7) and its ligand, mucosal addressin cell adhesion molecule-1, on high endothelial venules represents a key adhesion event during lymphocyte homing to secondary lymphoid tissue. Stromal cell-derived factor-1alpha (SDF-1alpha) is a chemokine that attracts T and B lymphocytes and has been hypothesized to be involved in lymphocyte homing. In this work we show that alpha(4)beta(7)-mediated adhesion of CD4(+) T lymphocytes and the RPMI 8866 cell line to mucosal addressin cell adhesion molecule-1 was up-regulated by SDF-1alpha in both static adhesion and cell detachment under shear stress assays. Both naive and memory phenotype CD4(+) T cells were targets of SDF-1alpha-triggered increased adhesion. In addition, SDF-1alpha augmented alpha(4)beta(7)-dependent adhesion of RPMI 8866 cells to connecting segment-1 of fibronectin. While pertussis toxin totally blocked chemotaxis of CD4(+) and RPMI 8866 cells to SDF-1alpha, enhanced alpha(4)beta(7)-dependent adhesion triggered by this chemokine was partially inhibited, indicating the participation of Galpha(i)-dependent as well as Galpha(i)-independent signaling. Accordingly, we show that SDF-1alpha induced a rapid and transient association between its receptor CXCR4 and Galpha(i), whereas association of pertussis toxin-insensitive Galpha(13) with CXCR4 was slower and of a lesser extent. SDF-1alpha also activated the small GTPases RhoA and Rac1, and inhibition of RhoA activation reduced the up-regulation of alpha(4)beta(7)-mediated lymphocyte adhesion in response to SDF-1alpha, suggesting that activation of RhoA could play an important role in the enhanced adhesion. These data indicate that up-regulation by SDF-1alpha of lymphocyte adhesion mediated by alpha(4)beta(7) could contribute to lymphocyte homing to secondary lymphoid tissues.