972. P27kip1 -Loaded Nanoparticle for the Inhibition of Intimal Hyperplasia in Mice Vein Grafting Model

clones) was amplified, and approximately 4 x 10"clones were randomly infused into HVJ-E vector. which were transferred to human aortic endothelial cells (HAEC). This assay identified three of the genes that were superior to VEG F in terms ofc-fos promoter activity. One gene in particular showed marked HAEC proliferative activity and c-fos promoter activity in comparison to VEGF and was used for subsequent experiments. Further study on this clone revealed that 96 bp segment is the active site of the clone. Thus, 30-amino acid peptide (AG-30) that derived from the segment was synthesized, and it showed strong angiogenic inducibility in further analysis, such as in vitro tube formation and in vivo matrigel plug assy, In addition, the angiogenic effect ofAG-30 in vivo, we developed slow release system ofAG-30 utilizing biodegradable gelatin microspheres.In the ischemic mouse hindlimb, the angiogenic score was improved in the slow-release AG-30-treated group in which blood flow was evaluated by laser Doppler imaging and capillary density was evaluated by immunostaining with anti-CD31 antibody. Interestingly, AG-30 was prospected having antimicrobial effect because of its a-helix structure with a number of hydrophobic or net positively charged amino acids and the propensity to fold into amphipathic structures. Indeed, AG-30 exhibited antimicrobial activity against Eaeruginosa, E.coli, and Siaureus. Overall , the current studies utilizing HVJ-E for high throughput screening resulted in successful isolating candidates for therapeutic gene, and the novel antimicrobial peptide, AG-30, may have therapeutic potential toward ischemic diseases, wound healing, and infectious diseases.

972. P27kip1-Loaded Nanoparticle for the Inhibition of Intimal Hyperplasia in Mice Vein Grafting Model Jing Ynag,' Cunxian Song,' Xiaoou Lang.' 'Institue ofBiomedical Engineering, Key Lab ofBiomaterial Research ofTianjin, Chinese Academy ofMedical Sciences, Tianjin, China; ]Vasclliar Surgery, First Clinical Hospital, China Medical University, Shenyang, China. Cardiovascular gene therapy has been hampered by a lack of effect gene delivery system. Intravascular gene therapy has been facilitated by gene-loaded nanoparticles (NPs) than can be delivered through an endovaseular diffusion catheter. It has been found that gene-loaded NPs increased the amount of gene entering cells and prevented the gene from degradation, therefore providing long-lasting biological effects. P27kipl is a member of cyclin-dependent kinase inhibitor family and it is known to prevent unrestrained cell proliferation. In the present study, a plasmid encoding the p27kipl was encapsulated into poly (lactic-co-glycolic acid) (PLGA) NPs by an emulsification / solvent evaporation technique. The NP size distribution was assessed by submicro laser defractometer. The particle morphology was observed by scanning electron microscopy. In vitro gene release from the NP was performed in TE buffer at37° under rotation (130 rpm) utilizing double-chamber diffusion cells on a shaking stand. Vein graft model was established in 120 rats by transplanting internal branch ofjugular vein to carotid artery. The rats were randomly divided into three groups: I) p27kip-NP treated grafting group; 2) control group (treated with empty NP without p27kipl gene); 3) shame grafting group. The grafted veins were harvested at 3d, 7d, 14d and 28d respectively after the operation. Intimal hyperplasic (IH) was observed by Morphologic evaluation. The expression of PCNA and E2F were detected by immunohistochemistry and analyzed by computer digitizing system. Results: The diameter ofp27kip I gene-NP was around 200 nm with vel)' narrow size distribution. The p27kip l-Ioaded NPs showed regular spherical shape with smooth surface. The p27kiplloading in NPs was about 3%. Encapsulation efficiency of gene was abou t 86%. The result of in vitro gene release showed that gene release from the NP lasted above two weeks. The p27kip I gene transfection mediated by NP Molecular Therapy Volume15. Supplement I. ~ br 2007 Copyright © The Ameri can Soci ety o r Gene "1l1f:r:lpy

vector resulted in increased protein expression ofp27kip I gene and there was a significant decrease in the intimal average thickness at 7, 14 and 28 days in p27kip I-NP treated group (P < 0.0 I), suggesting the intimal hyperplasia was inhibited. Furthermore. the intimal thickness decreased with time(6.124mm at 7ays, 2.477mm at 14days, 2.987mm at 28days)after single implantation of the p27kipl-NPs. There was no obvious difference between the 2 control groups. Immunohistochemical analysis of PCNA demonstrated that the number of positive cells in the p27kip I-NP group was much lesser compared with the control groups (p
973. Protective Effects of Limited In Vivo Gene Transfer of Phospholamban Antisense in a Catecholamine Induced Hypertrophic Rat Heart Model Motoki Sato, I Peter O'Gara, I Stephen J. Fuller, I Sian E. Harding.' 'Cardiac Medicine , National Heart and Lung Institute, Imperial College London. London, United Kingdom. Increase in SERCA2a activity, by decreasing the inhibitory protein, phospholamban, has been shown to rescue various models of hypertrophy and heart failure. In the present study we have used direct injection of adenovirus to transfect limited number of cells within the heart with antisense to phospholamban (PLBas), to determine whether individual rnyocytes can be protected in a diseased heart and whether such a protective effect influences on the global function. Transfected myocytes were identified after enzymativ isolation by co-expression of GFp, and contraction characteristics measured. Adenovirus with GFP alone was used as control. Hypertrophy was induced by chronic (14d) infusion ofnoradrenaline (NA) using osmotic minipumps, with saline in control groups (Sal). The heart was clipped during injection to confine adenovirus to the lower half: this produced limited transfection in the heart, with transfected as a percentage oftotal rnyocytcs being: GFP-Sal, 2.2±0.9%; GFPNA, 2.9± 1.8%; PLBas-Sal, 2.2±0.6%; PLBas-NA, 2.5± 1.5%. NA significantly increased the heart weight/tibial index from 3.21 ± 0.09 (GFP-Sal) to 3.96 ± 0.17 (GFP-NA) (p
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