Morphology and transfection study of human microvascular endothelial cell angiogenesis: an in vitro three-dimensional model.

Microvascular endothelial cells from human neonatal foreskin were grown in vitro until a three-dimensional network of capillary-like structures was formed. All stages of the angiogenic cascade could be observed in this in vitro model, including the formation of an internal lumen. The microscopy focused on morphology, formation of an internal lumen, role of the extracellular matrix, polarity of the cells, and the time-course of the angiogenic cascade. Bright-field microscopy revealed cells arranged circularly side by side and the internal lumen of capillary-like structures was verified by electron microscopy. Immunolabeling revealed a peritubular localization of collagen IV. Reporter gene expression after the formation of capillary-like structures was marginally higher than control expression, but clearly lower than the expression of cells at the stage of proliferation. Highest transfection efficiencies were obtained using vectors with the CMV promoter and the long fragment of the Ets-1 promoter. This is a first study of transfection efficiencies mapped for stages of in vitro angiogenesis. We describe here the morphological features of a long-term in vitro model of angiogenesis of human microvascular endothelial cells that could be used for transfection studies, without the provision of an extracellular matrix substrate. The cells self-create their own extracellular matrix to proliferate and form a three-dimensional network of capillary-like structures with an internal lumen.

Endostatin cDNA/cationic liposome complexes as a promising therapy to prevent lung metastases in osteosarcoma: study in a human-like rat orthotopic tumor.

Antiangiogenesis or destruction of tumor neovessels is an effective strategy to prevent tumor growth. Endostatin, one of the many inhibitors of angiogenesis that have been discovered, has shown conflicting results in preclinical assays. We studied the therapeutic potential of lipid/DNA complexes consisting of cationic liposomes and an endostatin-coding plasmid (Endo cDNA/CLP) in an orthotopic osteosarcoma model in rats. Empty plasmid without the endostatin gene complexed with cationic liposomes served as control. Animals were treated intravenously three times a week starting on the day tumors were detectable by (18)FDG tomoscintigraphy. During treatment, tumor progression was followed by PET scan and angioscintigraphy, and the effects of antivascular therapy on primary tumor, metastases, and tumor vascular density were confirmed by histologic analysis. Our results demonstrate that therapy using Endo cDNA/CLP is associated with pronounced delay in tumor growth. Moreover, it effectively prevented the occurrence of lung metastases, the major reason for bad prognosis and death in osteosarcoma patients. This approach could be used as an adjuvant therapy for osteosarcoma.

Targeting diphtheria toxin A-chain transcription to activated endothelial cells using an E-selectin promoter.

Targeting the transcription of a toxin gene to activated endothelial cells might be used for inhibiting angiogenesis in solid tumors. As a model, we transiently transfected human endothelial cells (HUVEC) in culture with expression plasmids for the toxic A-chain of diphtheria toxin (DT-A), using electroporation (achieving approximately 70% transfection efficiency). Protein synthesis in HUVEC was highly sensitive to DT-A expression from constitutive viral promoters. E-selectin is strongly expressed on HUVEC activated by TNFalpha or TPA. We therefore tested a human E-selectin promoter (-547 to +33) for targeting transcription of DT-A or reporter genes to HUVEC. Luciferase reporters were efficiently expressed in HUVEC from this promoter, with or without an enhancer responsive to Ets-1. Expression was increased by TNF alpha or TPA. DT-A showed highly preferential expression (increased by TNF alpha or TPA) in HUVEC, compared with WI38 human fibroblasts. HUVEC expressing DT-A were killed via apoptosis. Overall expression levels were influenced by alternative 'backbone' sequences used in the expression plasmids. We propose that delivery of transcriptionally regulated expression plasmids for DT-A in vivo, using cationic lipids that show preferential accumulation in activated or proliferating endothelium, may offer a novel means of inhibiting undesired angiogenesis.