PO06 | Extracellular vesicles profiles in patients with porto-sinusoidal vascular disease

S. Toffanin¹, E. Campello¹, A. Zanetto², M.C. Radu¹, E. Pinto², K. Fernande², C. Bulato¹, B. Serena¹, L. Spiezia¹, P. Burra², M. Senzolo², P. Simioni¹ | ¹First Chair of Internal Medicine, Department of Medicine, University Hospital of Padua Medical School, Padua; ²Department of Surgery Oncology and Gastroenterology, Gastroenterology and Multivisceral Transplant Unit, University Hospital of Padua, Italy

Background and Aims: Porto-sinusoidal vascular disorder (PSVD) has recently been proposed to delineate a group of hepatic vascular diseases characterized by lesions involving the portal venules and sinusoids, irrespective of the presence/absence of portal hypertension. Although data is still limited, several hypotheses and emerging evidence suggest that extracellular vesicles (EVs) might exert a functional role in the pathogenesis of PSVD. The analysis of EVs in PSVD may unveil disease-specific alterations in intercellular communication, contributing to understand the prothrombotic mechanisms and to discover novel biomarkers for diagnosis and prognosis. The study aims: i) to compare plasma-derived EVs in PSVD patients vs. healthy controls by flow cytometry; ii) to evaluate their potential involvement in disease progression.

Methods: Twenty-nine PSVD patients (median age 58 yrs; 20 males, 9 females) were included and compared with 9 (4 males and 5 females) aged matched (±3 yrs) healthy controls. Large extracellular vesicles (L-EVs) were isolated from platelet-poor plasma by centrifugation at 14,000 g for 30 min at 4°C, immunolabeled with calcein-AM, annexin V, CD41, CD62P, CD45, CD14, CD62E, anti-human-tissue factor (TF), CD105 and CD147. For EV size calibration, fluorescent polystyrene beads Gigamix were used to set a gate between 0.2 and 0.9 μm bead populations, defined as L-EVs gate. EVs were expressed as events/μl (absolute count) with the volume measurement of the CytoFLEX S.

Results: All enrolled patients had histologically confirmed PSVD. Among them, 12 had unprovoked PSVD, 4 were drug-related, 3 were associated with gastrointestinal diseases, 3 with immune-mediated conditions, and 7 cases were linked to myeloproliferative disorders. Endothelium-derived L-EVs co-expressing calcein-AM, annexin V, CD62E, and TF were significantly increased in PSVD patients compared to healthy controls (p<0.0001), reflecting endothelial activation and prothrombotic predisposition (Table 1). Patients also exhibited a marked increase in platelet-derived L-EVs, particularly calcein-AM+/annexin V+/CD41+/CD62P+ L-EVs (p=0.004), indicating enhanced platelet activation and a contribution to vascular and coagulative responses (Table 1). Notably, endothelial and platelet L-EV significantly correlated with PSVD etiology (r=0.38, p=0.04), with patients affected by myeloproliferative disease–related PSVD showing significantly higher levels than those with other etiologies (p=0.046 and 0.032, respectively). Angiogenesis-related calcein-AM+/annexin V+/CD105+/CD147+ L-EVs were significantly elevated in patients compared to controls (p<0.0001), suggesting active vascular remodeling. Regarding the inflammatory panel, patients showed significantly lower levels of calcein-AM+/annexin V+/CD45+/CD14+ L-EVs compared to controls (p=0.0002), indicating reduced monocyte/leukocyte-driven inflammatory activation (Table 1).

Conclusions: In conclusion, patients with PSVD display a L-EV profile characterized by vascular remodeling, endothelial and platelet activation. On the other hand, patients exhibited significantly lower levels of inflammatory L-EVs, suggesting limited systemic immune activation. Our preliminary findings support the hypothesis that PSVD is primarily driven by endothelial dysfunction and platelet activation, rather than by leukocyte-mediated inflammation. The etiology of PSVD appears to influence L-EV levels.