RESUMEN
UV light-based damage to skin cells can cause photoaging and skin cancer. A major cause of UV light-induced damage to skin is increased free radicals, such as superoxides. Increased superoxides can cause oxidative and nitrative damage to cell components. Thus, agents that counteract these damages may have therapeutic value. Herein, we show that angiopoietin-1 (ang1) prevented and blocked H(2)O(2)-induced increases in superoxides in human spontaneously immortalized keratinocyte line, HaCaT, and primary melanocytes (HeMn). Ang1 prevented H(2)O(2)-induced increases in damage to DNA (8-hydroxy-2'-deoxyguanosine) and proteins (nitrotyrosinylation). Ang1 promoted skin cell metabolism/viability, adhesion, and akt and MAPK(p42/44) activations. Using multi-gene transcriptional profiling, we found that skin cells express integrin subunits {(beta(1), beta(4-6), beta(8), alpha(v), alpha(2), alpha(3), alpha(6) (HaCaT)), (beta(1), beta(3), beta(5), beta(8), alpha(v), alpha(3) (HeMn))} and lack tie2 receptor mRNA. Integrin antibodies (alpha(v), beta(1)) disrupted skin cell adhesion to ang1 and ang1-induced decreases in superoxides. Our findings show that ang1 blocks free radical damage to skin cells and may be clinically useful to prevent and/or reduce photoaging and skin cancer.
Asunto(s)
Angiopoyetina 1/farmacología , Queratinocitos/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Envejecimiento de la Piel/efectos de los fármacos , Angiopoyetina 1/metabolismo , Adhesión Celular/efectos de los fármacos , Adhesión Celular/fisiología , Línea Celular Transformada , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/fisiología , Daño del ADN/efectos de los fármacos , Perfilación de la Expresión Génica , Humanos , Peróxido de Hidrógeno/metabolismo , Peróxido de Hidrógeno/farmacología , Integrinas/genética , Queratinocitos/citología , Queratinocitos/metabolismo , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Sistema de Señalización de MAP Quinasas/fisiología , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Oxidantes/metabolismo , Oxidantes/farmacología , Estrés Oxidativo/fisiología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Piel/citología , Piel/metabolismo , Envejecimiento de la Piel/fisiología , Neoplasias Cutáneas/metabolismo , Neoplasias Cutáneas/prevención & control , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
Angiostatin, a proteolytic fragment of plasminogen, is a potent endogenous antiangiogenic agent. The molecular mechanisms governing angiostatin's antiangiogenic and antitumor effects are not well understood. Here, we report the identification of mitochondrial compartment as the ultimate target of angiostatin. After internalization of angiostatin into the cell, at least 2 proteins within the mitochondria bind this molecule: malate dehydrogenase, a member of Krebs cycle, and adenosine triphosphate synthase. In vitro and in vivo studies revealed differential regulation of key prosurvival and angiogenesis-related proteins in angiostatin-treated tumors and tumor-endothelium. Angiostatin induced apoptosis via down-regulation of mitochondrial BCL-2. Angiostatin treatment led to down-regulation of c-Myc and elevated levels of another key antiangiogenic protein, thrombospondin-1, reinforcing its antitumor and antiangiogenic effects. Further evidence is provided for reduced recruitment and infiltration of bone marrow-derived macrophages in angiostatin-treated tumors. The observed effects of angiostatin were restricted to the tumor site and were not observed in other major organs of the mice, indicating unique tumor specific bioavailability. Together, our data suggest mitochondria as a novel target for antiangiogenic therapy and provide mechanistic insights to the antiangiogenic and antitumor effects of angiostatin.
Asunto(s)
Inhibidores de la Angiogénesis/farmacología , Angiostatinas/fisiología , Apoptosis , Regulación de la Expresión Génica , Mitocondrias/metabolismo , Animales , Línea Celular Tumoral , Ensayo de Inmunoadsorción Enzimática , Humanos , Masculino , Ratones , Ratones SCID , Modelos Biológicos , Neoplasias/terapia , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
Angiopoietins were thought to be endothelial cell-specific via the tie2 receptor. We showed that angiopoietin-1 (ang1) also interacts with integrins on cardiac myocytes (CMs) to increase survival. Because ang1 monomers bind and activate integrins (not tie2), we determined their function in vivo. We examined monomer and multimer expressions during physiological and pathological cardiac remodeling and overexpressed ang1 monomers in phenylephrine-induced cardiac hypertrophy. Cardiac ang1 levels (mRNA, protein) increased during postnatal development and decreased with phenylephrine-induced cardiac hypertrophy, whereas tie2 phosphorylations were unchanged. We found that most or all of the changes during cardiac remodeling were in monomers, offering an explanation for unchanged tie2 activity. Heart tissue contains abundant ang1 monomers and few multimers (Western blotting). We generated plasmids that produce ang1 monomers (ang1-256), injected them into mice, and confirmed cardiac expression (immunohistochemistry, RT-PCR). Ang1 monomers localize to CMs, smooth muscle cells, and endothelial cells. In phenylephrine-induced cardiac hypertrophy, ang1-256 reduced left ventricle (LV)/tibia ratios, fetal gene expressions (atrial and brain natriuretic peptides, skeletal actin, beta-myosin heavy chain), and fibrosis (collagen III), and increased LV prosurvival signaling (akt, MAPK(p42/44)), and AMPK(T172). However, tie2 phosphorylations were unchanged. Ang1-256 increased integrin-linked kinase, a key regulator of integrin signaling and cardiac health. Collectively, these results suggest a role for ang1 monomers in cardiac remodeling.
Asunto(s)
Angiopoyetina 1/química , Angiopoyetina 1/metabolismo , Cardiomegalia/prevención & control , Integrinas/metabolismo , Angiopoyetina 1/genética , Animales , Secuencia de Bases , Cardiomegalia/inducido químicamente , Cardiomegalia/genética , Cardiomegalia/metabolismo , Línea Celular , Cartilla de ADN/genética , Células Endoteliales/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , Miocitos Cardíacos/metabolismo , Fenilefrina/toxicidad , Fosforilación , Unión Proteica , Proteínas Serina-Treonina Quinasas/metabolismo , Estructura Cuaternaria de Proteína , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptor TIE-2/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Remodelación Ventricular/genética , Remodelación Ventricular/fisiologíaRESUMEN
PURPOSE: The half-life of the antiangiogenic molecule endostatin that has been used in clinical trial is short ( approximately 2 h). In addition, approximately 50% of the clinical grade endostatin molecules lack four amino acids at their NH(2) termini. Lack of these amino acids gives rise to a molecule that is devoid of zinc, resulting in no antitumor activity. Our goal was to develop a new version of endostatin that does not show such deficiency. EXPERIMENTAL DESIGN: A recombinant human endostatin conjugated to the Fc domain of IgG was constructed and expressed in mammalian cell culture. The presence of Fc has been shown by previous investigators to play a major role in increasing the half-life of the molecule. Fc-endostatin was tested in tumor-bearing mice, and its half-life was compared with the clinical grade endostatin. RESULTS: The antitumor dose of Fc-endostatin was found to be approximately 100 times less than the clinical grade endostatin. The half-life of Fc-endostatin in the circulation was found to be weeks rather than hours, as observed for endostatin alone. In addition, a U-shaped curve was observed for antitumor activity of endostatin as a function of endostatin concentration delivered to the animals. CONCLUSION: Fc-endostatin is a superior molecule to the original clinical endostatin. Due to its long half-life, the amount of protein required is substantially reduced compared with the clinically tested endostatin. Furthermore, in view of the U-shaped curve of efficacy observed for endostatin, we estimate that the requirement for Fc-endostatin is approximately 700-fold less than endostatin alone. The half-life of endostatin is similar to that of vascular endothelial growth factor-Trap and Avastin, two other antiangiogenic reagents. We conclude that a new clinical trial of endostatin, incorporating Fc, may benefit cancer patients.
Asunto(s)
Endostatinas/inmunología , Endostatinas/farmacocinética , Fragmentos Fc de Inmunoglobulinas/inmunología , Inmunoglobulina G/inmunología , Melanoma Experimental/metabolismo , Neoplasias Pancreáticas/metabolismo , Proteínas Recombinantes de Fusión/farmacocinética , Animales , Apoptosis , Ensayo de Inmunoadsorción Enzimática , Semivida , Humanos , Técnicas para Inmunoenzimas , Etiquetado Corte-Fin in Situ , Masculino , Melanoma Experimental/patología , Ratones , Ratones Endogámicos C57BL , Ratones SCID , Mutación/genética , Neoplasias Pancreáticas/patología , Proteínas Recombinantes/uso terapéutico , Células Tumorales Cultivadas , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Many sight-threatening diseases have two critical phases, vessel loss followed by hypoxia-driven destructive neovascularization. These diseases include retinopathy of prematurity and diabetic retinopathy, leading causes of blindness in childhood and middle age affecting over 4 million people in the United States. We studied the influence of omega-3- and omega-6-polyunsaturated fatty acids (PUFAs) on vascular loss, vascular regrowth after injury, and hypoxia-induced pathological neovascularization in a mouse model of oxygen-induced retinopathy. We show that increasing omega-3-PUFA tissue levels by dietary or genetic means decreased the avascular area of the retina by increasing vessel regrowth after injury, thereby reducing the hypoxic stimulus for neovascularization. The bioactive omega-3-PUFA-derived mediators neuroprotectinD1, resolvinD1 and resolvinE1 also potently protected against neovascularization. The protective effect of omega-3-PUFAs and their bioactive metabolites was mediated, in part, through suppression of tumor necrosis factor-alpha. This inflammatory cytokine was found in a subset of microglia that was closely associated with retinal vessels. These findings indicate that increasing the sources of omega-3-PUFA or their bioactive products reduces pathological angiogenesis. Western diets are often deficient in omega-3-PUFA, and premature infants lack the important transfer from the mother to the infant of omega-3-PUFA that normally occurs in the third trimester of pregnancy. Supplementing omega-3-PUFA intake may be of benefit in preventing retinopathy.