Mahajan, V.; Kumar, A.; Jacob, J.; Constantine, M.; Richardson,, L. S.; Urrabaz-Garza, R.; Amabebe, E.; Tantengco, O. A.; Kammala, A. K.; Menon, R.

Background: Low-dose aspirin (LDA) reduces preeclampsia (PE) risk by up to 40%, yet its molecular effects on chorion trophoblast cells (CTCs) a fetal membrane lineage at the feto-maternal interface remain obscure. CTCs form a structural and immunoregulatory barrier whose dysfunction drives inflammation-associated membrane pathology in PE. Extracellular vesicles (EVs) released by CTCs may encode cellular stress and adaptation states, offering a molecular window into aspirin's timing-dependent effects on PE risk modification. Methods: Human CTCs were challenged with cigarette smoke extract (CSE) to model oxidative stress-driven PE pathology. Two paradigms were tested: (1) prophylactic aspirin (4 and 40 ug/ml) before and/or flanking CSE, and (2) therapeutic aspirin after CSE challenge. EVs were isolated via ultracentrifugation and size exclusion chromatography, characterized by nanoparticle tracking and immunoblotting, and profiled by quantitative mass spectrometry. Network pathway analysis and machine-learning biomarker selection defined EV-encoded molecular states. Results: CTC-derived EVs from CSE-exposed cells carried a PE-like proteomic signature marked by suppressed VEGF/ECM remodeling, activated TNF-p53 apoptotic signaling, and heightened inflammation. Prophylactic low-dose aspirin shifted EV cargo toward preserved angiogenic capacity (VEGFA, COL1A1, MMP14) with attenuated apoptotic and NF-kB signatures. High-dose aspirin produced broad transcriptional suppression without vascular benefit in EVs. Therapeutic aspirin partially rescued injury-associated EV cargo but failed to restore angiogenic signatures. Machine-learning analysis of EV proteomes identified a prophylactic biomarker panel anchored by HSPA8, SERPINF2, COL4A1, and PLOD1, linked to angiogenic recovery and redox balance. Conclusions: CTC-derived EV proteomic signatures capture dose and timing-dependent aspirin effects, positioning the chorion as a pharmacological "secondary responder" favoring cellular resilience over classical anti-inflammatory suppression. EV-based molecular profiling might offer a framework for stratifying aspirin responders from non-responders toward personalized PE prevention.