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BACKGROUND: Diabetes mellitus increases the risk of developing cardiovascular diseases and is associated with macro and microvascular complications. Various strategies to promote therapeutic angiogenesis in ischemic tissue have been investigated, including delivery of pro-angiogenic growth factors and the genes that encode them, but results have been disappointing. Risk factors, such as diabetes, may adversely impact the response to angiogenic strategies and limit efficacy in vivo. We sought to examine the revascularization effect of multiple pro-angiogenic genes on chronic hindlimb ischemia in both non-diabetic and diabetic mouse models. METHODS: Spontaneously type II diabetic male mice (db/db; with C57/Bl6 genetic Background) and their control (db/m) between 8and 10-week-old were studied. Unilateral hindlimb ischemia was induced in all animals by ligation and stripping of the left femoral artery. For gene delivery, plasmids containing pro-angiogenic genes were administrated intramuscularly directly into the ischemic distal hindlimb muscle, below the ligation site at 4 weeks (for early delivery) and at 6 weeks (for late delivery) after ligation procedure. Gene combinations included vascular endothelial growth factor (VEGF) and angiopoietin 1, 2 (Ang-1, 2). Relative blood flow to the foot was measured under standardized conditions by laser Doppler perfusion imaging (LDPI). Perfusion measurements were performed on postoperative days 1, 3, 7, week 2, 3, 4, 5, 6, 7 and 8. The perfusion ratio (occludedto-non-occluded leg) was calculated for each animal. Animals from all groups were sacrificed at various times after gene delivery for tissue sampling (PCR, westerns, immunohistochemistry). RESULTS: Diabetic mice showed reduced ability to induce angiogenesis following ischemia compared to non-diabetic mice. In response to VEGF gene therapy, non-diabetic mice responded with greater neovascularization compared to diabetic mice. Ang-1 mono-therapy promoted an angiogenic response in diabetic mice, but not in non-diabetic mice. Ang2 mono-therapy did not promote angiogenesis in either model. Temporally separated delivery of VEGF (early) and Ang-1 (late) promoted more neovascularization than any single gene therapy. Delivery of VEGF and Ang-2 (early) and Ang-1 (late) showed the greatest angiogenic response in both diabetic and non-diabetic mice. CONCLUSION: Diabetic mice show a reduced angiogenic response to VEGF and increased angiogenic response to Ang1 in compared to non-diabetic mice. Temporally delivery of VEGF, Ang-2 and Ang-1 gene combination resulted a greater perfusion recovery and a greater angiogenic response in ischemic hindlimb skeletal muscle, compared to single gene or multiple gene combinations without temporal separation in both diabetic and non-diabetic settings.

s S233 Canadian Society of Cardiac Surgeons (CSCS) Poster SURGERY POSTER SESSION II Sunday, October 26, 2014 349 ATG7-SILENCING INDUCES ENDOTHELIAL-MESENCHYMAL TRANSITION-LIKE PHENOTYPIC SWITCHING IN HUMAN UMBILICAL VEIN ENDOTHELIAL CELLS KK Singh, F Lovren, A Quan, Y Pan, H Teoh, S Verma

techniques: 1) single-layer running baseball stitch 2) doublelayer running baseball stitch 3) running horizontal mattress stitch combined with a baseball stitch. The aorta was pressurized with saline in a purpose-built apparatus which captured all leaked fluid for measurement of the leakage rate over time. The intra-aortic pressure was adjusted over 8 increments from 110 mmHg to supra-physiologic levels at 375 mmHg. Leakage rates were measured at each incremental pressure level and compared to previously measured baseline values. Eighteen trials were performed, with six trials in each of the three groups. RESULTS At all pressures, the horizontal mattress group was measured to have significantly less leakage when compared to single or double baseball stitch closure (p 0.05). The double layer baseball stitch shows a trend towards decreased leakage compared to the single layer closure. CONCLUSION This porcine model provides the first quantitative comparison of three commonly used aortotomy closure techniques. The running horizontal mattress stitch combined with a baseball stitch provides the greatest resistance to leakage throughout the normal, hypertensive, and supra-physiologic aortic pressures. This evidence suggests that this closure technique may provide superior hemostasis and resistance to rupture in the perioperative period.

Toronto, Ontario BACKGROUND: The molecular basis of bicuspid aortic valve development and susceptibility to early calcification remain poorly understood. MicroRNAs are essential post-transcriptional modulators of gene expression which coordinate and integrate multiple regulatory pathways involved in normal and abnormal development. We performed microRNA profiling to identify potential novel pathways differentially regulated in bicuspid vs. tricuspid aortic valves. METHODS & RESULTS: Aortic valve leaflets were obtained intraoperatively from patients with stenotic bicuspid (BAV) and tricuspid aortic valves (TAV) at the time of valve replacement (N 10 each). Total RNA extracted from some of the tissue samples was directly labeled with biotin and hybridized to the GenoExplorerTM microRNA (Human) Chip. This microarray contains 1583 human miRNA probes and provides a comprehensive and comparative profile analyses of miRNA expression Fluorescent signals were scanned with the GenePix® 4000B Microarray Scanner. The other tissue samples were either flash frozen for molecular analysis or fixed in formalin and sectioned for histology and histochemical analysis. Microarray analysis revealed 40 differentially expressed miRNA sequences in BAV vs. TAV samples. Nineteen miRNAs including miRNA-152, -373, -30e, and -145 were upregulated and amongst the 21 miRNAs that were down-regulated were miRNA-125b, -1260, -27a, and -141 (Figure). These findings were confirmed by quantitative RT-PCR. Notably, expression of miRNA-141, a known repressor of the master osteogenic transcription factor DLX-5 and a regulator of bone morphogenetic protein (BMP-2), was 14.9-folds lower in BAV vs. TAV. BAV leaflet tissues also demonstrated higher BMP2 expression compared to TAV tissues. CONCLUSION: We identify a novel miRNA profile in calcific BAV vs. TAV leaflet tissues. Specifically, marked downregulation of miRNA-141 in BAV may serve to regulate abnormal cell fate decisions and BMP-2-dependent calcification leading to altered susceptibility towards calcific aortic stenosis. 712 COMPLETE REVASCULARIZATION IS COMPROMISED IN OFFPUMP CORONARY ARTERY BYPASS GRAFTING

and echocardiographic follow-up. RESULTS: In-hospital mortality was 12%. One patient died in the operating room from atrioventricular groove rupture and 2 died from congestive heart failure postoperatively. During a mean follow-up of 51 31 months, 8 patients died. Of these, 3 were a result of cardiovascular causes. Four patients underwent reoperation because of failure of MV repair (2), endocarditis (1) or hemolysis (1). All survivors were NYHA 2, with none to trivial mitral regurgitation in all MV repairs (12). At 5 years, actuarial survival was 56 21%, freedom from cardiac death was 70 19% and freedom from MV reoperation was 82 16%. CONCLUSIONS: After aggressive decalcification of the posterior MV annulus both MV repair and replacement are feasible with acceptable procedure-related mortality. It is clear however, that decalcification increases complexity and risk. While an aggressive approach is advisable in young patients and those with infective endocarditis, the choice of a more conservative approach may be appropriate in fragile and elderly patients.