RESUMO
The trabecular meshwork (TM), an ocular tissue next to the cornea, is a major site for regulation of the aqueous humor outflow. Malfunctioning of this tissue is believed to be responsible for development of glaucoma, a major blinding disease. Myocilin is a gene directly linked to the most common form of glaucoma. Its protein product has been localized to both intra- and extra-cellular sites in TM cells. This study was to investigate the association of myocilin with mitochondria in TM cells. In vitro mitochondrial import assays showed that myocilin was imported to the TM mitochondria, targeting to mitochondrial membranes and/or the intermembrane space. The targeting was mediated mostly via the amino-terminal region of myocilin. When myocilin expression was induced either by treatment with dexamethasone or transfection with a myocilin construct, the mitochondrial membrane potential in TM cells, as assessed by JC-1 staining, was lowered. Subcellular fractionation and Western blot analyses confirmed that a portion of myocilin sedimented with the mitochondrial fractions. Upon anti-Fas treatment to provoke apoptosis, an increase of myocilin distribution in cytosolic fraction was observed, suggesting that myocilin was partially released from mitochondrial compartments. These results confirmed the association of myocilin with TM cell mitochondria and indicated that myocilin may have a proapoptotic role in TM cells.
Assuntos
Proteínas do Citoesqueleto/metabolismo , Proteínas do Olho/metabolismo , Glicoproteínas/metabolismo , Mitocôndrias/metabolismo , Malha Trabecular/citologia , Adolescente , Adulto , Western Blotting , Células Cultivadas , Pré-Escolar , Proteínas do Citoesqueleto/biossíntese , Proteínas do Citoesqueleto/química , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/farmacologia , Olho/citologia , Proteínas do Olho/biossíntese , Proteínas do Olho/química , Proteínas do Olho/genética , Proteínas do Olho/farmacologia , Fibroblastos/metabolismo , Glicoproteínas/biossíntese , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Pessoa de Meia-Idade , Membranas Mitocondriais/fisiologia , Peso Molecular , Porinas/biossíntese , Porinas/química , Frações Subcelulares/metabolismo , Radioisótopos de Enxofre/metabolismoRESUMO
Our previous studies have indicated that transforming growth factor (TGF)-beta1 and VEGF expression are increased in the smooth muscle cell (SMC) layer of the pulmonary vessels of lambs with pulmonary hypertension secondary to increased pulmonary blood flow. Furthermore, we found that TGF-beta1 expression increased before VEGF. Because of the increased blood flow in the shunt lambs, the SMC in the pulmonary vessels are exposed to increased levels of the mechanical force, cyclic stretch. Thus, in this study, using primary cultures of pulmonary arterial SMC isolated from pulmonary arteries of 4-wk-old lambs, we investigated the role of cyclic stretch in the apparent coordinated regulation of TGF-beta1 and VEGF. Our results demonstrated that cyclic stretch induced a significant increase in VEGF expression both at the mRNA and protein levels (P < 0.05). The increased VEGF mRNA was preceded by both an increased expression and secretion of TGF-beta1 and an increase in reactive oxygen species (ROS) generation. In addition, a neutralizing antibody against TGF-beta1 abolished the cyclic stretch-dependent increases in both superoxide generation and VEGF expression. Our data also demonstrated that cyclic stretch activated an NAD(P)H oxidase that was TGF-beta1 dependent and that NAD(P)H oxidase inhibitors abolished the cyclic stretch-dependent increase in VEGF expression. Therefore, our results indicate that cyclic stretch upregulates VEGF expression via the TGF-beta1-dependent activation of NAD(P)H oxidase and increased generation of ROS.
Assuntos
Miócitos de Músculo Liso/metabolismo , NADPH Oxidases/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , NADPH Oxidases/antagonistas & inibidores , Artéria Pulmonar/citologia , RNA Mensageiro/metabolismo , Ovinos , Estresse Mecânico , Fator de Crescimento Transformador beta1 , Fator A de Crescimento do Endotélio Vascular/genéticaRESUMO
The association of cytogenetic abnormalities with cancer is well established. However, due to the historic lack of specific insight into the functional role of these anomalies, they have mostly served as diagnostic and/or prognostic indicators. Recent developments in chronic myelogenous leukemia and breast cancer have raised hopes for specific cytogenetic alterations to serve as therapeutic targets. This article reviews the aid provided by molecular diagnostics in these exciting developments in the cancer arena.
Assuntos
Neoplasias da Mama/diagnóstico , Análise Citogenética , Leucemia Linfocítica Crônica de Células B/diagnóstico , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Neoplasias da Mama/genética , Neoplasias da Mama/terapia , Aberrações Cromossômicas , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/terapia , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapiaRESUMO
We have previously shown that TGF-1 and VEGF expression are increased in the smooth muscle cell (SMC) layer of the pulmonary vessels of lambs with pulmonary hypertension secondary to increased pulmonary blood flow. Further we found that TGF-1 expression increased prior to VEGF. Due to the increased blood flow in these lambs the SMC in the pulmonary vessels are exposed to increased levels of cyclic stretch. Thus, using primary cultures of pulmonary arterial SMCs (PASMCs) isolated from 4 week-old lambs, we investigated the role of cyclic stretch in the apparent coordinated regulation of TGF-1 and VEGF. Our results demonstrated that cyclic stretch induced a significant increase in VEGF expression both at the mRNA and protein preceded by an increase in expression and secretion of TGF-1 and an increase in reactive oxygen species (ROS) generation. In addition, a neutralizing TGF-1 antibody abolished the cyclic stretch-dependent increase in ROS and VEGF expression. Further, we found that cyclic stretch activated an NAD(P)H oxidase in a TGF-1 dependent manner that when inhibited abolished the cyclic stretch-dependent increase in VEGF expression. Our results indicate that cyclic stretch up-regulates VEGF expression via the TGF-1 dependent activation of NAD(P)H oxidase and increased generation of ROS.
