Cell Type-Specific Isolation of Extracellular Vesicles from Heart Failure Patient Plasma for Biomarker Discovery

Introduction: : Heart failure with preserved ejection fraction (HFpEF) represents a major disease target with its high prevalence and risk factors. However, early detection methods are lacking in number and efficacy. To address this obstacle, we investigated novel sources of biomarker candidates for the development of prognostic biomarkers, identification of disease phenotypes, and understanding of cell type-specific contributions to disease biology. These novel sources are cell type-specific subpopulations of extracellular vesicles (EVs) secreted into systemic blood circulation.

Materials and

Methods: : EVs are known to carry various biomolecules that can serve as biomarkers of disease and are actively studied in cancer and neurodegenerative disease fields. However, like HFpEF, EVs in blood are heterogeneous primarily because of their numerous tissue and cell type sources. Therefore, to perform shotgun proteomic analysis of EVs for biomarker identification, we used immunoprecipitation methods to isolate distinct EV subpopulations from relevant cell types to HFpEF. These include cardiac fibroblasts, endothelial cells, cardiomyocytes, neutrophils, and macrophages. We performed nanoparticle tracking analysis to measure EV size distribution, concentration, and surface charge for workflow validation. Cryogenic transmission electron microscopy was used to visualize EV morphology for isolation quality and purity assessment. We used super resolution microscopy to profile protein cargo on and within individual EVs to assess subpopulation purity, homogeneity, and confirm that isolated subpopulations were distinct. Lastly, we ran quantitative proteomics to identify differentially expressed proteins for biomarker identification as well as used gene ontology analysis to reveal protein signatures for disease biology elucidation.

Results, Conclusions, and Discussions:: Initial findings reveal detectable amounts of each EV subpopulation following sequential immunoprecipitation against cell type-specific EV surface markers. HFpEF EV subpopulations were compared to EVs from healthy control plasma via nanoparticle tracking analysis and no significant differences in size distribution, concentration, or EV surface charge was detected. EV subpopulations were imaged by cryogenic transmission electron microscopy and demonstrated typical morphology with a lipid bilayer membrane. Super resolution microscopy confirmed the expression of cell type-specific markers on EV surfaces and validated our immunoprecipitation protocol to have successfully distinct EV subpopulations for biomarker discovery. Quantitative proteomics identified over 1200 unique proteins and differential expression analysis revealed hundreds of protein biomarker candidates. The top ten biomarker candidates are currently being screened in a validation cohort. Gene ontology analysis and term enrichment analysis revealed disease associated proteins and protein signatures for each EV subpopulation, with many significantly associated pathways relevant to heart failure disease pathophysiology.
Altogether, we have established a methodology to fractionate plasma-derived EVs based on cell type of origin and will demonstrate the potential of these subpopulations for biomarker discovery in HFpEF patient samples.

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