| 권진숙, 정일하¹ , 김유경¹ , 박노관¹ , 배장환¹ , 황경국¹ , 김동운¹ , 고규영²,조명찬¹ |
Backgrounds and Objectives: COMP-Angiopoietin 1 (COMP-Ang 1) is a potent Ang 1 variant. Previously, we reported that direct injection of COMP-Ang 1 protein effectively attenuated ventricular remodeling by enhanced akt-survivin pathway in a rat model of myocardial infarction. However the mechanism of COMP-Ang 1 protein’s effect to the cardiomyocytes (CMCs) in the absence of Tie-2, receptor of Ang 1, was uncertain. We hypothesized that COMP-Ang 1 is able to be delivered directly, via a non-Tie-2 pathway, to the CMCs. Methods: To evaluate the effect of COMP-Ang 1 protein, neonatal rat CMCs were incubated under 2 different COMP-Ang 1 concentrations (350, 700 ng/ml) for 10, 20, 30 minutes at normoxic condition. COMP-Ang 1, including synthetic FLAG reporter protein, was pre-labeled with anti-FLAG FITC antibody. COMP Ang-1(+) cells were evaluated with anti- Troponin I (TnI), p-Akt, survivin antibody by immunocytochemistry, 3-D confocal image analysis. Quantitative analysis of COMP-1(+) cells was evaluated by FACS analysis. Results: 1) COMP Ang-1-FITC florescence particles were observed with intracytoplasmic scatter pattern in CMCs. Theses COMP Ang-1(+) cells were more observed under the condition of 700 ng/ml concentration and 30 minutes exposure. In immunocytochemistry with double staining using anti-TnI Texas red antibody, co-expression of green and red florescence were observed. The proportion of COMP Ang-1(+)/TnI (+) was approximately 2-3% at high power field. 2) The proportion of COMP Ang-1(+) cells by FACS analysis was 2.74±0.76 %. 3) And also, COMP Ang-1(+) cells strongly expressed p-Akt and survivin, known signal of survival pathway at confocal image analysis. Summaries and Conclusion: Although the precise mechansim of delivery is not elucidated in our study, endocytosis may be one of the possible mechanism of COMP Ang-1 protein delivery. COMP Ang-1 protein is directly delivered into the rat neonatal CMCs and enhances the expression of p-akt and survivin. This finding suggests COMP Ang-1 protein may be used as an effective tool for enhance CMCs’survival.
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