Soluble TNF-α receptor I encoded on plasmid vector and its application in experimental gene therapy of radiation-induced lung fibrosis.

Post-radiation inflammatory reaction leads to an irreversible pulmonary fibrosis which may cause lethal respiratory insufficiency. Pathological inflammatory and fibrotic changes might be attenuated by inhibiting tumour necrosis factor (TNF)-α activity using TNF-α soluble receptors. Thus, an experimental antifibrotic gene therapy with the plasmid vector encoding a mouse soluble receptor I for TNF-α (psTNFR-I) was assessed. Soluble TNFR-I encoding gene was cloned into pcDNA3.1 plasmid. The ability of psTNFR-I expressing vector to transfect cells, and its biological activity in vitro and in vivo were examined by PCR, RT-PCR, MTT assay and ELISA. The C57Bl/6J mice received single intramuscular injection of psTNFR-I, conjugated with polyetylenimine (PEI) 25 kDa, equally divided to both hind legs, 3 days before irradiation (20 Gy, Co60), and either a single injection or ten injections once a week after irradiation. The data proved the effectiveness of psTNFR-I product to neutralise TNF-α activity in vitro. The in vivo plasmid incorporation and maintenance was confirmed. Measurements of plasma soluble TNFR-I levels showed that the in vivo gene transfer was effective. PEI was found to enhance transfection efficiency in vivo. The psTNFR-I/PEI complexes caused no toxicity in the transfected mice. C57Bl/6J mice that received prolonged psTNFR-I/PEI injections developed lethal fibrotic syndrome and died 8 weeks later than the mice treated with a double plasmid injection and the control mice treated with a control plasmid. Sequential administration of soluble TNFR-I by a nonviral, intramuscular gene transduction in the early and late post-radiation inflammatory phase prolonged survival of irradiated mice and attenuated the symptoms of lung fibrosis. The psTNFR-I gene transduction may provide a safe and simple method to partially neutralise TNF-α activity and prevent radiation-induced lung injury.

Vascular endothelial growth factor and soluble FLT-1 receptor interactions and biological implications.

Vascular endothelial growth factor (VEGF), binding to an appropriate receptor like FLT, is the main mitogen for endothelial cells and a strong inducer of angiogenesis. A soluble form of VEGF receptor, sFLT-1, specifically binds VEGF and inhibits its activity. The following expression plasmids were used in the experiments: pVEGF plasmid encoding VEGF165, pFGF-2 encoding FGF-2 and psFLT-1 plasmid encoding the soluble form of VEGF receptor, sFLT-1. The interaction between VEGF and sFLT-1 was evaluated using a migration test and ERK1/2 activity utilizing mouse sarcoma cells (L-1). Implication of the VEGF/sFLT-1 action was also visualized using in vivo angiogenesis assay. The conditioned medium (CM) from L-1 phVEGF-165 transfectants stimulated L-1 cell migration more than medium from non-transfected L-1 cells. Media collected from phVEGF-165 transfectants or original L-1 cells only slightly stimulated the migration of cells transfected with psFLT-1. The L-1 cells also showed intensive phospho-ERK1/2 activity when treated with the CM from VEGF transfectants. In vivo tests showed that sFLT-1 effectively suppressed VEGF-mediated angiogenesis without affecting FGF-2-driven angiogenesis. To summarize, this study documented that sFLT-1 released from transfected cells might inhibit cell functions induced by VEGF, but not by FGF. The results obtained from in vivo angiogenesis tests also confirm the antiangiogenic potency of cloned sFLT-1, which can be useful for planning cancer experimental therapy studies.

Role of v-Raf and truncated form RAF1 in the induction of vascular endothelial growth factor and vascularization.

In this work we studied the effect of v-raf and different form RAF on up regulation of VEGF gene expression and angiogenesis. In the experiments the truncated forms of RAF1 gene were transfected into the cells of the established human urothelial line HCV-29, otherwise non-angiogenic. The plasmids coding for mitochondrial targeted dominant active or dominant negative RAF1 (DAM, DNM respectively) were deleted with RAS binding domain. DAM has the ability to phosphorylate BAD whereas DNM cannot phosphorylate BAD. DAM RAF1, similarly to v-raf transfected cells, induced up-regulation of VEGF and caused angiogenic phenotype as studied in vivo. DNM RAF1 transfectants induced VEGF independent angiogenesis.

The role of focal adhesion kinase in the emigration of cells from confluent cultures.

We studied the effect of the modification of focal adhesion kinase (FAK) on the growth, migration and adhesion of C3H 10T1/2 cells. Cells transfected with plasmid coding for antisense FAK displayed a low level of FAK protein. Interestingly, the transfected cells achieved a higher saturation density at confluence, and displayed reduced adhesion and enhanced emigration from a confluent layer of cells when stimulated with fibronectin. In conclusion, it can be postulated that FAK plays an important role in the mechanism of contact inhibition.