Dissociated expression of natural killer 1.1 and T-cell receptor by invariant natural killer T cells after interleukin-12 receptor and T-cell receptor signalling.

Invariant (i) natural killer T (NKT) cells become undetectable after stimulation with alpha-galactosylceramide (alpha-GalCer) or interleukin (IL)-12. Although down-modulation of surface T-cell receptor (TCR)/NKR-P1C (NK1.1) expression has been shown convincingly after stimulation with alpha-GalCer, it is unclear whether this also holds true for IL-12 stimulation. To determine whether failure to detect iNKT cells after IL-12 stimulation is caused by dissociation/internalization of TCR and/or NKR-P1C, or by block of de novo synthesis of these molecules, and to examine the role of IL-12 in the disappearance of iNKT cells after stimulation with alpha-GalCer, surface (s)/cytoplasmic (c) protein expression, as well as messenger RNA (mRNA) expression of TCR/NKR-P1C by iNKT cells after stimulation with alpha-GalCer or IL-12, and the influence of IL-12 neutralization on the down-modulation of sTCR/sNKR-P1C expression by iNKT cells after stimulation with alpha-GalCer were examined. The s/cTCR(+ )s/cNKR-P1C(+) iNKT cells became undetectable after in vivo administration of alpha-GalCer, which was partially prevented by IL-12 neutralization. Whereas s/cNKR-P1C(+) iNKT cells became undetectable after in vivo administration of IL-12, s/cTCR(+) iNKT cells were only marginally affected. mRNA expression of TCR/NKR-P1C remained unaffected by alpha-GalCer or IL-12 treatment, despite the down-modulation of cTCR and/or cNKR-P1C protein expression. By contrast, cTCR(+ )cNKR-P1C(+) sTCR(-) sNKR-P1C(-) iNKT cells and cNKR-P1C(+) sNKR-P1C(-) iNKT cells were detectable after in vitro stimulation with alpha-GalCer and IL-12, respectively. Our results indicate that TCR and NKR-P1C expression by iNKT cells is differentially regulated by signalling through TCR and IL-12R. They also suggest that IL-12 participates, in part, in the disappearance of iNKT cells after stimulation with alpha-GalCer by down-modulating not only sNKR-P1C, but also sTCR.

An efficient gene transfer method mediated by ultrasound and microbubbles into the kidney.

BACKGROUND: Safety issues are of paramount importance in clinical human gene therapy. From this point of view, it would be better to develop a novel non-viral efficient gene transfer method. Recently, it was reported that ultrasound exposure could induce cell membrane permeabilization and enhance gene expression. METHODS: In this study, we examined the potential of ultrasound for gene transfer into the kidney. First, we transfected rat left kidney with luciferase plasmid mixed with microbubbles, Optison, to optimize the conditions (duration of ultrasound and concentration of Optison). Then, 4, 7, 14 and 21 days after gene transfer, luciferase activity was measured. Next, localization of gene expression was assessed by measuring luciferase activity and green fluorescent protein (GFP) expression. Expression of GFP plasmid was examined under a fluorescence microscope at 4 and 14 days after gene transfer. Finally, to examine the side effects of this gene transfer method, biochemical assays for aspartate aminotransferase (AST), alanine aminotransferase (ALT), blood urea nitrogen (BUN) and creatinine (Cre) were performed. RESULTS: Optison and/or ultrasound significantly enhanced the efficiency of gene transfer and expression in the kidney. Especially, 70-80% of total glomeruli could be transfected. Also, a significant dose-dependent effect of Optison was observed as assessed by luciferase assay (Optison 25%: 12.5 x 10(5) relative light units (RLU)/g tissue; 50%: 31.3 x 10(5) RLU/g tissue; 100%: 57.9 x 10(5) RLU/g tissue). GFP expression could be observed in glomeruli, tubules and interstitial area. Results of blood tests did not change significantly after gene transfer. CONCLUSIONS: Overall, an ultrasound-mediated gene transfer method with Optison enhanced the efficiency of gene transfer and expression in the rat kidney. This novel non-viral method may be useful for gene therapy for renal disease.

Enhanced therapeutic angiogenesis by cotransfection of prostacyclin synthase gene or optimization of intramuscular injection of naked plasmid DNA.

BACKGROUND: Although clinical trials of therapeutic angiogenesis by angiogenic growth factors with intramuscular injection of naked plasmid DNA have been successful, there are still unresolved problems such as low transfection efficiency. From this viewpoint, we performed the following modifications: (1) combination with vasodilation using prostacyclin and (2) changing the agents or volume of naked plasmid DNA in vivo. METHODS AND RESULTS: First, we examined cotransfection of the VEGF gene with the prostacyclin synthase gene in a mouse hindlimb ischemia model. Cotransfection of the VEGF gene with the prostacyclin synthase gene resulted in a further increase in blood flow and capillary density compared with single VEGF gene. Similar results were obtained with other angiogenic growth factors, such as hepatocyte growth factor (HGF). Alternatively, we changed the injection volume of the solution of plasmid DNA. Luciferase activity was increased in a volume-dependent manner. An increase in injection volume at 1 site rather than separate injections at multiple sites resulted in high transfection efficiency, which suggests that transfection of naked plasmid DNA is mediated by pressure. Interestingly, treatment with hyperbaric oxygen increased the transfection efficiency. Finally, we also examined the effects of different solutions. Saline and PBS, but not water, achieved high transfection efficiency. In addition, sucrose solution but not glucose solution resulted in high luciferase activity. CONCLUSIONS: Overall, angiogenesis might be enhanced by cotransfection of prostacyclin synthase gene or an increase in injection volume and osmotic pressure. These data provide important information for the clinical application of therapeutic angiogenesis to treat peripheral arterial disease.

Angiogenesis induced by endothelial nitric oxide synthase gene through vascular endothelial growth factor expression in a rat hindlimb ischemia model.

BACKGROUND: Because the mechanism of the angiogenic property of nitric oxide (NO) was not fully understood in vivo, we focused on the role of vascular endothelial growth factor (VEGF) in angiogenesis induced by endothelial NO synthase (eNOS) gene transfer. METHODS AND RESULTS: After intramuscular injection of eNOS DNA into a rat ischemic hindlimb, transfection of eNOS vector resulted in a significant increase in eNOS protein 1 week after transfection. In addition, tissue concentrations of nitrite and nitrate were significantly increased in rats transfected with the eNOS gene up to 2 weeks after transfection. The increase in tissue nitrite and nitrate concentrations was completely inhibited by NG-nitro-L-arginine methyl ester (L-NAME). In contrast, serum concentrations of nitrite and nitrate and blood pressure were not changed by eNOS gene transfer. Importantly, overexpression of the eNOS gene resulted in a significant increase in peripheral blood flow, whereas L-NAME inhibited the increase in blood flow. Interestingly, basal blood flow was significantly lower in rats treated with L-NAME than in control rats. A significant increase in capillary number was consistently detected in rats transfected with the eNOS gene at 4 weeks after transfection, accompanied by a significant increase in VEGF. Moreover, administration of neutralizing anti-VEGF antibody abolished the increase in blood flow and capillary density induced by eNOS plasmid injection. CONCLUSIONS: Overall, intramuscular injection of bovine eNOS plasmid induced therapeutic angiogenesis in a rat ischemic hindlimb model, a potential therapy for peripheral arterial disease. The stimulation of angiogenesis by NO might be due to upregulation of local VEGF expression.

Angiogenic property of hepatocyte growth factor is dependent on upregulation of essential transcription factor for angiogenesis, ets-1.

BACKGROUND: Although hepatocyte growth factor (HGF) is an angiogenic growth factor, it is still unclear how it exerts its angiogenic effects. Thus, we focused on the role of an essential transcription factor for angiogenesis, ets-1. In this study, we addressed the following specific questions: (1) what genes responsible for angiogenesis can be regulated by HGF and (2) whether upregulation of gene expression for angiogenesis is dependent on ets-1. METHODS AND RESULTS: In human endothelial cells, HGF significantly stimulated the matrix-degrading pathway, such as the production of matrix metalloprotease-1 (MMP-1) through its specific receptor, c-met. In addition, HGF also significantly increased HGF itself and its specific receptor, c-met. Moreover, HGF significantly increased the transcription activity and mRNA expression of ets-1 in a time-dependent manner. Importantly, transfection of antisense ets-1 oligodeoxynucleotides (ODN) resulted in a significant reduction in MMP-1, HGF and c-met. Interestingly, HGF also stimulated ets-1 mRNA in vascular smooth muscle cells, similar to endothelial cells. Of importance, transfection of antisense ets-1 ODN resulted in a significant decrease in vascular endothelial growth factor (VEGF) and HGF expression, whereas HGF stimulated both HGF and VEGF expression. Moreover, in vivo transfection of ets-1 antisense ODN resulted in an inhibition of angiogenesis induced by the HGF gene in a rat ischemic hindlimb model. CONCLUSIONS: Here, we demonstrated that HGF stimulated the expression of MMP-1, VEGF, HGF itself, and c-met in human endothelial cells and vascular smooth muscle cells. Upregulation of angiogenesis-related genes was largely dependent on the induction of ets, especially ets-1. These data provide new information about the mechanisms of angiogenesis.

Enhanced angiogenesis and improvement of neuropathy by cotransfection of human hepatocyte growth factor and prostacyclin synthase gene.

The current therapeutic angiogenesis strategy to treat ischemic disease by using angiogenic growth factors has been limited to use of a single gene. However, as vasodilator substances such as prostacyclin are widely used for the treatment of peripheral arterial disease, it might be useful to combine angiogenesis with vasodilation of new vessels. In a mouse hind limb ischemia model, cotransfection of the hepatocyte growth factor (HGF) gene with the prostacyclin synthase gene demonstrated a further increase in blood flow and capillary density compared with a single gene. Even in the rabbit ischemia model, cotransfection of HGF plasmid with the prostacyclin synthase gene demonstrated a further increase in angiogenic activity compared with HGF alone. Because peripheral neuropathy due to diabetes is common for significant morbidity, we examined the hypothesis that experimental diabetic neuropathy can be reversed by HGF and prostacyclin synthase genes. Severe peripheral neuropathy, characterized by significant slowing of nerve conduction velocity compared with nondiabetic control animals, was ameliorated. Overall, cotransfection of the prostacyclin synthase and HGF genes is more effective than single-gene transfection to stimulate angiogenesis, and it significantly improved neuropathy. These data provide important information relating to the clinical application of therapeutic angiogenesis to treat peripheral arterial disease.

Therapeutic approach to familial hypercholesterolemia by HVJ-liposomes in LDL receptor knockout mouse.

Familial hypercholesterolemia (FH) is an inherited disease in humans, which we have used as a model to develop a new strategy of gene therapy. This disease, which is due to mutation in the low density lipoprotein (LDL) receptor gene and results in deficiency of the LDL receptor, is associated with hypercholesterolemia and premature development of coronary heart disease. This disease has been identified as one of the target diseases for gene therapy, because a 50% reduction of cholesterol level would be beneficial in such patients. In this study, we examined the feasibility of gene therapy by the delivery of the human LDL receptor plasmid into the liver via the portal vein. For gene transfer we utilized HVJ-liposome method with which many successful gene transfers have been reported. Administration of the human LDL receptor plasmid by the HVJ-liposome method into the liver resulted in a decrease of total cholesterol level. Moreover, second administration of this gene two weeks after the first administration resulted in sustained lowering of total cholesterol level. Although single administration of plasmid by the HVJ-liposome method induced antibodies against HVJ, this antibody production did not affect gene expression following second administration. These results suggest the possibility of a novel repetitive gene therapy for FH, using human LDL receptor plasmid transfer directly into the liver by the HVJ-liposome method.