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The Role of Exosomes in Cellular Communication in Both PAH Disease Progression and Therapeutic Success
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A2079 - The Role of Exosomes in Cellular Communication in Both PAH Disease Progression and Therapeutic Success
Author Block: R. Harper1, S. Maiolo1, R. Lim2, D. Greening3, M. Cockshell4, C. Bonder4, P. N. Reynolds1; 1Thoracic Medicine, Royal Adelaide Hospital, Adelaide, Australia, 2Monash Univ, Melbourne 3149 VIC, Australia, 3Dept. Biochemistry and Genetics, La Trobe University, Melbourne, Australia, 4Centre for Cancer Biology, Adelaide, Australia.
Introduction/Aim: Previously we reported the therapeutic effect of BMPR2 augmented endothelial progenitor cell therapy in a rat PH model. We know there is little direct cell integration into the pulmonary endothelium following intravenous injection, thus we attribute the positive physiological effects of our cell therapy to cell-to-cell communication via secreted factors such as exosomes (exos). In this study we look at comparing PAH-EPC and control-EPC protein profiles via mass spectrometry, and we assess their capacity to interact with and transport BMPR2 into endothelial cells (EC). Methods: EPCs are isolated and cultured from 15mL of PAH or control peripheral blood. Cells were either transduced with AdBMPR2 or AdTrackLuc, or untransduced for subsequent exo isolations. Exosomes are isolated via differential centrifugation, and characterised with a NanoSight, TEM and SEM. Exosome protein profile was via mass spectrometry. Exo-Quick™ treated exos and GFP-exos labelled were used to view exo localisation within the target ECs both live and fixed via confocal microscopy. BMPR2-Exos were incubated on ECs for 48hrs before being washed off, and the cells lysed for western blot analysis. Results: Exosomes were positively identified as 40-200nm via NanoSight, TEM and SEM. Protein profiling of exos showed differential protein expression in the PAH vs control for 326 proteins, including a relative down-regulation of BMPR2 in PAH exos. Exosome interaction with ECs demonstrated localisation around the nucleus of the cell, both in live and fixed EC samples. Additionally, BMPR2 expression was significantly increased in cells treated with BMPR2-Exos. Conclusion: Differential protein expression between exos derived from PAH compared to control EPCs was shown. Additionally, we have characterised exos interaction with ECs and shown that they are involved in BMPR2 transfer from BMPR2 transduced EPCs to naïve ECs. These results indicate the potential role of exos in our BMPR2-cell therapy.
Author Block: R. Harper1, S. Maiolo1, R. Lim2, D. Greening3, M. Cockshell4, C. Bonder4, P. N. Reynolds1; 1Thoracic Medicine, Royal Adelaide Hospital, Adelaide, Australia, 2Monash Univ, Melbourne 3149 VIC, Australia, 3Dept. Biochemistry and Genetics, La Trobe University, Melbourne, Australia, 4Centre for Cancer Biology, Adelaide, Australia.
Introduction/Aim: Previously we reported the therapeutic effect of BMPR2 augmented endothelial progenitor cell therapy in a rat PH model. We know there is little direct cell integration into the pulmonary endothelium following intravenous injection, thus we attribute the positive physiological effects of our cell therapy to cell-to-cell communication via secreted factors such as exosomes (exos). In this study we look at comparing PAH-EPC and control-EPC protein profiles via mass spectrometry, and we assess their capacity to interact with and transport BMPR2 into endothelial cells (EC). Methods: EPCs are isolated and cultured from 15mL of PAH or control peripheral blood. Cells were either transduced with AdBMPR2 or AdTrackLuc, or untransduced for subsequent exo isolations. Exosomes are isolated via differential centrifugation, and characterised with a NanoSight, TEM and SEM. Exosome protein profile was via mass spectrometry. Exo-Quick™ treated exos and GFP-exos labelled were used to view exo localisation within the target ECs both live and fixed via confocal microscopy. BMPR2-Exos were incubated on ECs for 48hrs before being washed off, and the cells lysed for western blot analysis. Results: Exosomes were positively identified as 40-200nm via NanoSight, TEM and SEM. Protein profiling of exos showed differential protein expression in the PAH vs control for 326 proteins, including a relative down-regulation of BMPR2 in PAH exos. Exosome interaction with ECs demonstrated localisation around the nucleus of the cell, both in live and fixed EC samples. Additionally, BMPR2 expression was significantly increased in cells treated with BMPR2-Exos. Conclusion: Differential protein expression between exos derived from PAH compared to control EPCs was shown. Additionally, we have characterised exos interaction with ECs and shown that they are involved in BMPR2 transfer from BMPR2 transduced EPCs to naïve ECs. These results indicate the potential role of exos in our BMPR2-cell therapy.
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