RESUMEN
OBJECTIVE: To evaluate the effect of angiopoietin-1, an angiogenic growth factor, on lung capillary leakage and survival in a murine model of acute lung injury. DESIGN: Laboratory investigation. SETTING: Research laboratory at New York University School of Medicine and Department of Veterans Affairs, NY Harbor Healthcare System. SUBJECTS: C57BL/6 mice weighing 18-20 g, susceptible to endotoxin-induced acute lung injury. INTERVENTIONS: Acute lung injury was induced in C57BL/6 mice by the intraperitoneal administration of endotoxin. The effects of angiopoietin-1, expressed from a nonreplicating E1a-deleted adenovirus containing the angiopoietin-1 complementary DNA (AdAng1), on survival and lung injury were evaluated. An E1a-deleted adenovirus that does not contain a transgene (Ad312) and phosphate-buffered saline were used as controls. MEASUREMENTS AND MAIN RESULTS: Angiopoietin-1 protein was detected by immunoblotting in the serum of mice that received an intraperitoneal injection of AdAng1 but not in mice that received the control virus Ad312. When compared with control groups, mice that received AdAng1 5 days before endotoxin administration had improved survival and significantly less protein leakage from the circulation into the lungs, as detected by quantitative spectrophotometric measurements of Evans blue dye. Furthermore, when compared with controls, histopathology and immunostaining of lungs against CD31 and smooth muscle actin suggested preservation of vascular integrity and decreased tissue damage in mice pretreated with AdAng1. When endotoxin administration preceded infection with AdAng1 by 3 hrs, no benefit was observed. CONCLUSIONS: These data show that adenoviral mediated expression of angiopoietin-1 can protect against the development of lung capillary protein leak and decrease the mortality induced by endotoxin. However, the timing of AdAng1 administration in relation to the onset of lung injury may be critical.
Asunto(s)
Angiopoyetina 1/uso terapéutico , Edema Pulmonar/tratamiento farmacológico , Angiopoyetina 1/sangre , Animales , Modelos Animales de Enfermedad , Endotoxinas , Inyecciones Intraperitoneales , Lipopolisacáridos , Ratones , Ratones Endogámicos C57BL , Edema Pulmonar/etiología , Edema Pulmonar/patología , Valores de Referencia , Resultado del TratamientoRESUMEN
Replicating adenoviral vectors are capable of multiplying up to a thousandfold in the target cell, a property that might prove to be of tremendous potential for cancer therapy. However, restricting viral replication and toxicity to cancer cells is essential to optimize safety. It has been proposed that modifications of the E1a protein that impair binding to Rb or p300 will prevent S-phase induction in normal cells, resulting in selective viral replication in tumor cells. However, it remains uncertain which of the several possible E1a modifications would be most effective at protecting normal cells without compromising the oncolytic effect of the vector. In this study, we have expressed several E1a-deletion mutants at high levels using the CMV promoter and tested them for their ability to facilitate S-phase induction, viral replication, and cytotoxicity in both normal and cancer cells. Deletion of the Rb-binding domain within E1a only slightly decreased the ability of the virus to induce S phase in growth-arrested cells. The effect of this deletion on viral replication and cytotoxicity was variable. There was reduced cytotoxicity in normal bronchial epithelial cells; however, in some normal cell types there was equal viral replication and cytotoxicity compared with wild type. Deletions in both the N-terminus and the Rb-binding domain were required to block S-phase induction effectively in growth-arrested normal cells; in addition, this virus demonstrated reduced viral replication and cytotoxicity in normal cells. An equally favorable replication and cytotoxicity profile was induced by a virus expressing E1a that is incapable of binding to the transcriptional adapter motif (TRAM) of p300. All viruses were equally cytotoxic to cancer cells compared with wild-type virus. In conclusion, deletion of the Rb-binding site alone within E1a may not be the most efficacious means of targeting viral replication and toxicity. However, deletion within the N-terminus in conjunction with a deletion within the Rb-binding domain, or deletion of the p300-TRAM binding domain, induces a more favorable cytotoxicity profile.
Asunto(s)
Adenoviridae/genética , Proteínas E1A de Adenovirus/genética , Terapia Genética/métodos , Vectores Genéticos/toxicidad , Neoplasias/terapia , Replicación Viral/genética , Proteínas E1A de Adenovirus/metabolismo , Secuencias de Aminoácidos/genética , Sitios de Unión/genética , Línea Celular Tumoral , Humanos , Inmunoprecipitación , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína/genética , Proteína de Retinoblastoma/genética , Proteína de Retinoblastoma/metabolismo , Fase S , Eliminación de Secuencia , Transactivadores/metabolismoRESUMEN
Oncolytic replicating adenoviruses are a promising new modality for the treatment of cancer. Despite the assumed biologic advantage of continued viral replication and spread from infected to uninfected cancer cells, early clinical trials demonstrate that the efficacy of current vectors is limited. In xenograft tumor models using immune-incompetent mice, wild-type adenovirus is also rarely able to eradicate established tumors. This suggests that innate immune mechanisms may clear the virus or that barriers within the tumor prevent viral spread. The aim of this study was to evaluate the kinetics of viral distribution and spread after intratumoral injection of virus in a human tumor xenograft model. After intratumoral injection of wild-type virus, high levels of titratable virus persisted within the xenograft tumors for at least 8 weeks. Virus distribution within the tumors as determined by immunohistochemistry was patchy, and virus-infected cells appeared to be flanked by tumor necrosis and connective tissue. The close proximity of virus-infected cells to the tumor-supporting structure, which is of murine origin, was clearly demonstrated using a DNA probe that specifically hybridizes to the B1 murine DNA repeat. Importantly, although virus was cleared from the circulation 6 hr after intratumoral injection, after 4 weeks systemic spread of virus was detected. In addition, vessels of infected tumors were surrounded by necrosis and an advancing rim of virus-infected tumor cells, suggesting reinfection of the xenograft tumor through the vasculature. These data suggest that human adenoviral spread within tumor xenografts is impaired by murine tumor-supporting structures. In addition, there is evidence for continued viral replication within the tumor, with subsequent systemic dissemination and reinfection of tumors via the tumor vasculature. Despite the limitations of immune-incompetent models, an understanding of the interactions between the virus and the tumor-bearing host is important in the design of effective therapies.
Asunto(s)
Adenoviridae/genética , Vectores Genéticos/farmacocinética , Neoplasias Experimentales/metabolismo , Adenoviridae/fisiología , Animales , Línea Celular Transformada , Tejido Conectivo/metabolismo , Sondas de ADN , Terapia Genética , Vectores Genéticos/administración & dosificación , Vectores Genéticos/sangre , Humanos , Inmunohistoquímica , Inyecciones , Cinética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones , Ratones Desnudos , Neoplasias Experimentales/patología , Neoplasias Experimentales/terapia , Células Tumorales Cultivadas , Replicación Viral , Ensayos Antitumor por Modelo de Xenoinjerto , Factor de von Willebrand/análisis , Factor de von Willebrand/inmunologíaRESUMEN
Gene transfer of p53 induces cell death in most cancer cells, and replication-defective adenoviral vectors expressing p53 are being evaluated in clinical trials. However, low transduction efficiency limits the efficacy of replication-defective vector systems for cancer therapy. The use of replication-competent vectors for gene delivery may have several advantages, holding the potential to multiply and spread the therapeutic agent after infection of only a few cells. However, expression of a transgene may adversely affect viral replication. We have constructed a replicating adenoviral vector (Adp53rc) that expresses high levels of p53 at a late time point in the viral life cycle and also contains a deletion of the adenoviral death protein (ADP). Adp53rc-infected cancer cells demonstrated high levels of p53 expression in parallel with the late expression pattern of the adenoviral fiber protein. p53 expression late in the viral life cycle did not impair effective virus propagation. Survival of several lung cancer cell lines was significantly diminished after infection with Adp53rc, compared with an identical p53-negative control virus. p53 expression also improved virus release and spread. Interestingly, p53 was more cytotoxic than the ADP in cancer cells but less cytotoxic than the ADP in normal cells. In conclusion, late expression of p53 from a replicating virus improves tumor cell killing and viral spread without impairing viral replication. In addition, in combination with a deletion of the ADP, specificity of tumor cell killing is improved.