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Mandy Turner, PhD




Research Fellow








Mandy E Turner PhD1, Mark C Blaser1, Cassandra L Clift PhD1, Shiori Kuraoka PhD1, Tan Pham BS1, Jochen D Muehlschlegel MD MMSc2, Tsuyoshi Kaneko MD3, Sasha A Singh PhD1, Masanori Aikawa, MD, PhD1,4, Elena Aikawa MD PhD1,4

Optimization of tissue resident extracellular vesicle isolation from fibrocalcific aortic valves

I am a Research Fellow at the Center for Interdisciplinary Cardiovascular Sciences at BWH under the supervision of Dr. Elena Aikawa. Using “omics” techniques and molecular and extracellular vesicle approaches, I am examining extracellular vesicle-mediated cell crosstalk in vascular calcification and the impact of chronic kidney disease on the pathophysiology calcified aortic valve disease. I hope to identify therapeutic targets to address this unmet clinical need. This symposium enables the growth of collaborative groups of diverse women who sponsor and mentor each other, and I hope to contribute to this environment and learn from exceptional leaders and peers.

Background: Matrix-bound extracellular vesicles (EVs) are key local mediators of cardiovascular calcification. However, isolation and characterization of EVs from fibrocalcific tissue is challenging. Here, we sought to establish a streamlined methodology to isolate tissue EVs from frozen specimens.

Methods: To isolate EVs, frozen calcified human aortic valves underwent collagenase digestion, serial ultra-centrifugation, and density gradient fractionation. The protocol was halted and frozen at three centrifugation steps and compared to fresh-frozen tissue with no stoppage (N=2/group). Isolations were characterized using transmission electron miscopy (TEM), nanoparticle tracking analysis (NTA), and proteomics.

Results: EVs isolated from later freezing stop-points had more particles of EV-like size (2.56×1011±1.03 x1011 vs 2.36×1012±1.34 x1011, p<0.001). TEM visualized different morphological and contaminant profiles at each stop-point, including collagen and lipid particles. Freezing later in the isolation protocol improved the similarity of proteins identified in the samples (57% vs 80%) compared to frozen control. Confirmatory EV signature was characterized by the identification of 14 key vesicular cargo and surface markers.

Conclusions: EVs that were isolated after freezing later in the protocol had the most similarity to fresh frozen tissue. Optimization of this protocol will facilitate the integration of EV isolation from fibrocalcific tissues and enable ex vivo EV studies.