Hypoxia-driven complement activation expands circulating endothelial cells in transplant-associated thrombotic microangiopathy: A multi-omics single-cell and functional study

Abstract

Abstract Background Transplant-associated thrombotic microangiopathy (TA-TMA) lacks validated biomarkers and targeted therapies. We hypothesized that peri-transplant hypoxia activates complement and drives the emergence of pathogenic circulating endothelial cells (CECs). Methods In a prospective, multi-center cohort we analyzed peripheral-blood 10× Genomics scRNA-seq libraries from 10 TA-TMA patients and 10 kidney-transplant controls. CECs were quantified by multiparameter flow cytometry (CD45^–CD31^+CD146^+). Differential expression, Gene Set Enrichment Analysis, and CellChat ligand-receptor modeling assessed pathway activation and cellular crosstalk. Functional validation used HUVECs exposed to 1 % O₂ ± siHIF1A, eculizumab, or roxadustat, with read-outs of C3b deposition, trans-endothelial electrical resistance (TEER), and detachment. Mixed-effects and Cox models correlated molecular metrics with clinical outcomes. Results Clinical/phenotypic: Median onset of TA-TMA was 48 days post-transplant. CECs comprised 10.8 % (IQR 8.9–13.4) of circulating mononuclear cells versus 1.9 % (1.2–2.6) in controls (5.7-fold↑, p < 0.0001). Transcriptomic: CECs from TA-TMA displayed robust enrichment of the HIF-1α pathway (NES 2.4, FDR 1×10⁻⁴); complement genes C3, CFB, CFD were co-up-regulated (log₂FC 1.8–2.3, FDR < 0.01). CellChat revealed heightened VEGFA–KDR and C3aR1 signaling from inflammatory monocytes to CECs (interaction score ↑3.1-fold). Biomarker correlations: Hypoxia score strongly correlated with plasma sC5b-9 (Spearman r = 0.72, p = 0.003) and 4-week eGFR decline (r = –0.65, p = 0.01). Baseline CEC > 5 % and high hypoxia score independently predicted 90-day graft loss (HR 4.3, 95 % CI 1.4–13.2, p = 0.01). Functional assays: 1 % O₂ increased C3b deposition on HUVECs 2.6 ± 0.4-fold vs. normoxia (p = 0.002) and reduced TEER 38 ± 6 %. Both siHIF1A and eculizumab halved C3b loading and restored barrier integrity; roxadustat (HIF-PHD inhibitor) phenocopied hypoxia-induced complement activation. Conclusions Hypoxia activates complement and fuels CEC expansion in TA-TMA, linking two key pathogenic drivers. CEC percentage and a hypoxia transcriptomic score emerge as non-invasive early biomarkers, while combined hypoxia- and complement-targeted interventions warrant clinical testing to improve allograft outcomes.