Elevated TMAO is Associated with Pulmonary Arterial Endothelial Cell Pyroptosis and Sumoylation-Related Caspase-1 Signaling in MCT-Induced Pulmonary Arterial Hypertension Rats

Introduction: Pulmonary Arterial Hypertension (PAH) is characterized by progressive pulmonary vascular remodeling, in which Pulmonary Arterial Endothelial Cell (PAEC) dysfunction and inflammation play important roles. The gut microbiota-derived metabolite trimethylamine N-oxide (TMAO) has been associated with PAH and vascular inflammation, but the relationship between TMAO, PAEC pyroptosis, and post-translational regulation of Caspase-1 in PAH remains incompletely understood. The association between elevated TMAO and PAEC pyroptosis and SUMOylation-related Caspase-1 signaling in monocrotaline (MCT)-induced PAH was investigated. Methods: PAH was induced in rats by a single intraperitoneal injection of MCT (60 mg/kg). Hemodynamic parameters, right ventricular hypertrophy, and pulmonary vascular remodeling were assessed. Plasma TMAO, IL-1β, and IL-18 levels were measured, whereas GSDMD, Caspase-1, and SUMO-1 expression in CD31-positive pulmonary arterial endothelial cells was examined by immunofluorescence. Potential SUMOylation sites on Caspase-1 were predicted using SUMOplot, JASSA, and GPS-SUMO. 3,3-Dimethyl- 1-butanol (DMB), a microbial TMA production inhibitor, was used to reduce circulating TMAO levels. Results: MCT-induced PAH was accompanied by increased plasma TMAO levels, pulmonary vascular remodeling, right ventricular hypertrophy, increased GSDMD expression in CD31-positive PAECs, and elevated circulating IL-1β and IL-18. Bioinformatic analyses identified several putative SUMOylation sites on Caspase-1, with K256 showing a high prediction score. SUMO-1 and Caspase-1 expression were also increased in PAECs from MCT-treated rats. DMB treatment reduced circulating TMAO levels and attenuated hemodynamic abnormalities, right ventricular hypertrophy, and pulmonary vascular remodeling. These changes were accompanied by reduced GSDMD, SUMO-1, and Caspase-1 expression and lower plasma IL-1β and IL-18 levels. Discussion: Elevated circulating TMAO in MCT-induced PAH was accompanied by increased GSDMD, SUMO-1, and Caspase-1 expression in PAECs and elevated plasma IL-1β and IL-18 levels, whereas reduction of TMAO by DMB attenuated these molecular changes together with pulmonary vascular remodeling. These findings extend the previously recognized association between TMAO and PAH by linking elevated TMAO to pyroptosis-associated alterations in the pulmonary arterial endothelium. The coordinated changes in SUMO-1 and Caspase-1, together with the bioinformatic prediction of potential SUMOylation sites on Caspase-1, further suggest the possible involvement of SUMOylation-related Caspase-1 signaling. However, direct SUMO conjugation of Caspase-1 and modification of the predicted K256 site were not experimentally demonstrated; therefore, this regulatory mechanism remains a hypothesis requiring further validation. Conclusion: Elevated TMAO is associated with enhanced PAEC pyroptosis-related signaling and coordinated increases in SUMO-1 and Caspase-1 expression in MCT-induced PAH. Reducing circulating TMAO with DMB attenuates these molecular abnormalities, pulmonary hemodynamic alterations, right ventricular hypertrophy, and pulmonary vascular remodeling. These findings support a potential link between TMAO and SUMOylation-related Caspase-1 signaling in PAH and identify this pathway as a candidate mechanism for future investigation.