Cationic lipid-mediated gene transfer of cystic fibrosis transmembrane conductance regulator (CFTR) cDNA represents a promising approach for treatment of cystic fibrosis (CF). Here, we report on the structures of several novel cationic lipids that are effective for gene delivery to the lungs of mice. An amphiphile (#67) consisting of a cholesterol anchor linked to a spermine headgroup in a "T-shape" configuration was shown to be particularly efficacious. An optimized formulation of #67 and plasmid vector encoding chloramphenicol acetyl-transferase (CAT) was capable of generating up to 1 microgram of CAT enzyme/lung following intranasal instillation into BALB/c mice. This represents a 1,000-fold increase in expression above that obtained in animals instilled with naked pDNA alone and is greater than 100-fold more active than cationic lipids used previously for CFTR gene expression. When directly compared with adenovirus-based vectors containing similar transcription units, the number of molecules of gene product expressed using lipid-mediated transfer was equivalent to vector administration at multiplicities of infection ranging from 1 to 20. The level of transgene expression in the lungs of BALB/c mice peaked between days 1 and 4 post-instillation, followed by a rapid decline to approximately 20% of the maximal value by day 7. Undiminished levels of transgene expression in the lung could be obtained following repeated intranasal administration of #67:DOPE:pCF1-CAT in nude mice. Transfection of cells with formulations of #67:DOPE:pCF1-CFTR generated cAMP-stimulated CFTR chloride channel and fluid transport activities, two well-characterized defects associated with CF cells. Taken together, the data demonstrate that cationic lipid-mediated gene delivery and expression of CFTR in CF lungs is a viable and promising approach for treatment of the disease.

Abstract-Hypoxia-inducible factor-1 (HIF-1) mediates transcriptional activation of vascular endothelial growth factor (VEGF) and other hypoxia-responsive genes. Transgenic expression of a constitutively stable HIF-1␣ mutant increases the number of vascular vessels without vascular leakage, tissue edema, or inflammation. This study aimed to investigate the molecular basis by which HIF-1 mediates the angiogenic response to hypoxia. In primary human endothelial cells, hypoxia, desferrioxamine, or infection with Ad2/HIF-1␣/VP16, an adenoviral vector encoding a constitutively stable hybrid form of HIF-1␣, increased the mRNA and protein levels of VEGF, angiopoietin-2 (Ang-2), and angiopoietin-4 (Ang-4). Infection with Ad2/CMVEV (a control vector expressing no transgene) had no effect. Angiopoietin-1 (Ang-1) expression was not detected in human endothelial cells. Ang-4 was also induced by hypoxia or Ad2/HIF-1␣/VP16 in human cardiac cells, whereas Ang-1 expression remained unchanged. Recombinant Ang-4 protein protected endothelial cells against serum starvation-induced apoptosis and increased cultured endothelial cell migration and tube formation. Ad2/HIF-1␣/VP16 stimulated endothelial cell proliferation and tube formation. Hypoxia-or Ad2/HIF-1␣/VP16-induced tube formation was significantly reduced by a Tie-2 inhibitor. These results suggest that HIF-1 mediates the angiogenic response to hypoxia by upregulating the expression of multiple angiogenic factors. Ang-4 can function similarly as Ang-1 and substitute for Ang-1 to participate in hypoxia-induced angiogenesis. Activation of the angiopoietin/Tie-2 system may play a role in the ability of HIF-1 to induce hypervascularity without excessive permeability. (Circ Res. 2003;93:664-673.)Key Words: hypoxia-inducible factor-1 Ⅲ hypoxia Ⅲ angiogenesis Ⅲ angiopoietins Ⅲ vascular endothelial growth factor A dministration of a single growth factor in the form of protein or gene has been shown to promote tissue neovascularization in animal models and patients. 1,2 However, transgenic overexpression of vascular endothelial growth factor (VEGF) alone in mice results in increased numbers of primarily leaky vascular vessels with tissue edema and inflammation, 3,4 suggesting that VEGF needs to work in conjunction with other angiogenic factors to produce a healthy vasculature. 5,6 In a variety of conditions, such as malignant tumors, wound healing, and myocardial ischemia, hypoxia is a fundamental stimulus for angiogenesis. 7,8 Activation of hypoxia-responsive genes including VEGF is mediated by hypoxia-inducible factor-1 (HIF-1), a heterodimeric basic helix-loop-helix-PAS domain transcription factor. 9,10 HIF-1 is composed of two subunits, HIF-1␣ and HIF-1␤ (aryl hydrocarbon nuclear translocator). Whereas the ␤-subunit protein is constitutively present, the stability of the ␣-subunit and its transcriptional activity are precisely controlled by the intracellular oxygen concentration. [11][12][13][14][15] Knocking-out the HIF-1␣ alleles in mice results in embryonic lethality with vascul...

1 We assessed the relaxant effect of 17f,-oestradiol (10 -, 10-6 and 10-M) on rabbit isolated coronary arteries precontracted with prostaglandin F2,, (3 x 10-6M), high extracellular potassium (30mM) and Bay K 8644 (10-6M) plus high extracellular potassium (15 mM) by measuring isometric tension. 17fl-Oestradiol (10-6 and 10-5M) induced significant relaxation in coronary arteries from male and female rabbits. No differences were seen between arteries with or without endothelium. There were also' no differences between coronary arteries isolated from male and female rabbits. 2 Inhibitors of endothelium-derived relaxing factor and vasodilator prostanoids, namely, reduced haemoglobin, Nw-nitro-L-arginine methyl ester and indomethacin, did not affect the relaxation induced by 17fl-oestradiol in endothelium-intact coronary arteries.3 Methylene blue, an inhibitor of guanylate cyclase, did not affect the coronary artery relaxation induced by 17/1-oestradiol. 4 The' calcium concentration-dependent contraction curve in potassium-depolarization medium was shifted to the right by 17fl-oestradiol (10-6 and 10-5M) in the rabbit coronary artery and rat aorta. The -log EC50 s of calcium in control and after incubation with 17fi-oestradiol (10-6 and 10 -M) were 3.7 + 0.09, 3.1 + 0.10 and 2.8 + 0.08 respectively in rabbit coronary arteries and 3.8 + 0.11, 3.3 + 0.14 and 2.9 + 0.15 in rat aorta. 5 The results indicate that 17fi-oestradiol induces rabbit coronary artery relaxation by an endotheliumindependent mechanism in vitro. A calcium antagonistic property may be involved in the mechanism of rabbit coronary arterial relaxation by 17fi-oestradiol.

The HIF-1alpha/VP16 hybrid transcription factor is able to promote significant improvement in perfusion of an ischemic limb. These results confirm the feasibility of a novel approach for therapeutic angiogenesis in which neovascularization may be achieved indirectly by use of a transcriptional regulatory strategy.