Proteome Profiling of Extra-cellular Vesicles in Pulmonary Arterial Hypertension

Rationale: National Survey on Drug Use and Health in 2023 reported lifetime methamphetamine (MA) use among 5.7% Americans, with MA causing 2.64-fold more PAH(Pulmonary Arterial Hypertension)-related hospitalizations compared to non-users. Mechanisms behind cause-and-effect relationship between MA and PAH is not yet established. Methods: So, in this study, we did proteome profiling of circulating extracellular vesicles (EVs)from MAPAH(Methamphetamine-PAH)(n=8), and Idiopathic PAH(IPAH)(n=11) patients enrolled at University of Kansas Health System with the long-term goal of identifying unique mechanisms and highly sensitive & stable biomarkers of MAPAH. Results: Median age of IPAH and MAPAH patients included for the analysis was 64 and 54 years, respectively. Other demographic difference between two groups were that MAPH patients were all white while IPAH predominantly constituted females. Clinically, MAPAH patients relatively had worse hemodynamic parameters compared to IPAH [mPAP(53.75 ± 10.44 vs 50.9 ± 19.15 mm of Hg), PVR(11.36 ± 5.619 vs 9.209 ± 6.815 wood unit), and arterial oxygen saturation(96 ± 2.708 vs 98.38 ± 1.685%)]. Proteomic profiling of plasma derived small EVs using Olink®HT explore proximity extension assay revealed multiple altered EV proteins (see volcano plot A) in PAH patients when compared with normal control individuals(n=10) including enrichment of cell surface interactions at vascular wall and neutrophil degranulation pathway in PAH. Specifically higher levels of EV-linked. Complement 9 in PAH patients showed positive correlation with mPAP and PVR. Further analysis of altered proteins using Ingenuity pathway Analysis(IPA)predicted Pulmonary Hypertension as top end point (p < 1.32e-6), reinforcing relevance of our findings. To identify unique cargo in MAPAH-EVs we compared MAPAH and IPAH groups. Levels of IL31,"itchy” cytokine, that is known to increase smooth muscle cell contraction and TNF Receptor-Associated Factor 3 Interacting Protein 1 involved in immune regulation and autophagy were elevated in MA-PAH EVs ( Volcano plot B). Furthermore, KEGG and IPA analysis of altered protein cargo showed decrease in the proteins related to immune cell movement, hematopoiesis, and growth factors, alongside enrichment of death receptor signaling, cytokine-cytokine interaction pathway, autophagy and apoptosis and reduced wound healing suggesting higher organismal death in MA-PAH group. Conclusion: Overall, our preliminary study has identified unique and common EV proteins in MAPAH and IPAH and these plasma derived EVs can be further studied for better elucidation of disease mechanisms and to identify unique biomarkers.