RESUMO
The class II α-isoform of phosphatidylinositol 3-kinase (PI3K-C2α) is localized in endosomes, the trans-Golgi network and clathrin-coated vesicles; however, its functional role is not well understood. Global or endothelial-cell-specific deficiency of PI3K-C2α resulted in embryonic lethality caused by defects in sprouting angiogenesis and vascular maturation. PI3K-C2α knockdown in endothelial cells resulted in a decrease in the number of PI3-phosphate-enriched endosomes, impaired endosomal trafficking, defective delivery of VE-cadherin to endothelial cell junctions and defective junction assembly. PI3K-C2α knockdown also impaired endothelial cell signaling, including vascular endothelial growth factor receptor internalization and endosomal RhoA activation. Together, the effects of PI3K-C2α knockdown led to defective endothelial cell migration, proliferation, tube formation and barrier integrity. Endothelial PI3K-C2α deficiency in vivo suppressed postischemic and tumor angiogenesis and diminished vascular barrier function with a greatly augmented susceptibility to anaphylaxis and a higher incidence of dissecting aortic aneurysm formation in response to angiotensin II infusion. Thus, PI3K-C2α has a crucial role in vascular formation and barrier integrity and represents a new therapeutic target for vascular disease.
Assuntos
Barreira Alveolocapilar/metabolismo , Neovascularização Fisiológica , Fosfatidilinositol 3-Quinases/metabolismo , Angiotensina II/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Caderinas/genética , Caderinas/metabolismo , Adesão Celular , Movimento Celular/genética , Proliferação de Células , Células Cultivadas , Vesículas Revestidas por Clatrina/metabolismo , Células Endoteliais da Veia Umbilical Humana , Humanos , Camundongos , Camundongos Knockout , Fosfatidilinositol 3-Quinases/deficiência , Fosfatidilinositol 3-Quinases/genética , Interferência de RNA , RNA Interferente Pequeno , Receptores de Fatores de Crescimento do Endotélio Vascular/metabolismo , Transdução de Sinais/genética , Proteína rhoA de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismo , Rede trans-Golgi/metabolismoRESUMO
We report here a tumor-targeting strategy for pancreatic cancer using a modified auxotrophic strain of Salmonella typhimurium. The genetically-modified strain of S. typhimurium requires the amino acids arginine and leucine. These mutations preclude growth in normal tissue but do not reduce bacterial virulence in tumor cells. The tumor-targeting strain of S. typhimurium, termed A1-R and expressing green fluorescent protein (GFP), was administered to an orthotopic human pancreatic tumor expressing red fluorescent protein (RFP) in nude mice. After 7 days of treatment, the pancreatic cancer had regressed without the need of chemotherapy or any other treatment. This new strategy demonstrates the clinical potential of bacterial targeting for pancreatic cancer.
Assuntos
Neoplasias Pancreáticas/microbiologia , Neoplasias Pancreáticas/terapia , Salmonella typhimurium/fisiologia , Animais , Arginina/metabolismo , Linhagem Celular Tumoral , Proteínas de Fluorescência Verde/genética , Humanos , Leucina/metabolismo , Camundongos , Camundongos Nus , Mutação/genética , Organismos Geneticamente Modificados , Transfecção , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
As previously shown, the stem cell marker nestin is expressed in nascent blood vessels in transgenic nestin-driven green fluorescent protein (ND-GFP) nude mice. This mouse model was recently utilized to evaluate angiogenesis in primary tumors in an orthotopic model of pancreatic cancer. In the present study, nascent angiogenesis of pancreatic cancer liver metastasis in the ND-GFP transgenic nude mice after splenic injection of low-passage xPA-1 human pancreatic cancer cells expressing red fluorescent protein (RFP) was visualized by dual-color fluorescence imaging. ND-GFP was highly expressed in proliferating endothelial cells and nascent blood vessels in the growing liver metastasis. Immunohistochemical staining showed that CD31 co-localized in ND-GFP-expressing nascent blood vessels. The density of nascent blood vessels in the tumor was readily quantitated. Gemcitabine significantly decreased the mean nascent blood vessel density in the pancreatic liver metastases. In conclusion, the dual-color model of the ND-GFP nude mouse with RFP-expressing pancreatic cancer liver metastases, enabled the simultaneous visualization and quantitation of nascent angiogenesis and its response to angiogenesis inhibitors. This model will be useful for understanding the mechanism of angiogenesis of pancreatic cancer liver metastasis and for the discovery of effective new inhibitors of this process.