NLRP3 inhibitor Dapansutrile (OLT1177) Protects Against Trimethylamine N-oxide (TMAO) induced Endothelial Dysfunction

Background: Trimethylamine N-oxide (TMAO), a metabolite produced by gut microbiota is known to cause a plethora of cardio-metabolic diseases. We have shown earlier that TMAO induced endothelial injury by activation of NOD-like receptor protein 3 (NLRP3) inflammasomes. This study aims to evaluate the effect of NLRP3 inhibitor, Dapansutrile (OLT1177), on TMAO-induced activation of NLRP3 pathway and resultant endothelial injury. Methods: Endothelial (EOMA) cells were cultured and treated with various concentrations of OLT1177 (1µM, 5µM, 10 µM and 15 µM) (n=4/group) to assess the cell viability. EOMA cells were seeded on trans well membranes at a density of 1x10 5 and the integrity of endothelial cell monolayer was evaluated by FITC-dextran after treatment with TMAO (60µM) and OLT1177 (10µM) (n=6/group) for 24h. The gene expression pattern of NLRP3, Caspase, ASC, ZO1 and VE-cadherin was assessed in response to TMAO with and without OLT1177 treatment using RT-qPCR analysis. Junctional protein expression (ZO1 and VE-cadherin) was assessed by immunoblotting and immunofluorescent staining. Results: OLT1177 treatment at (1µM, 5µM, 10 µM and 15 µM) concentrations did not induce any changes in the cell viability of EOMA cells indicating that the OLT1177 can be safely used on endothelial cells up to 15 µM concentrations. We observed a significant increase in the relative permeability of endothelial cell monolayers upon TMAO treatment which was comparatively lower in cells treated with NLRP3 inhibitor OLT1177 (p<0.05). In response to TMAO treatment, expression pattern of ZO1 and VE-cadherin was downregulated whereas caspase, ASC and NLRP3 gene expression was seen to be upregulated. OLT1177 treatment significantly decreased the transcript levels of Caspase, ASC, NLRP3 demonstrating inhibition of the NLRP3 inflammasome pathway. In addition, OLT1177 treatment restored ZO1 and VE-cadherin gene expression. Western blot analysis demonstrated that TMAO treatment significantly decreased the expression of tight junctional proteins ZO1 and VE-cadherin but not in the OLT1177 treated group. Immunofluorescence results revealed that TMAO caused disruption in these tight junctional proteins which were restored upon OLT1177 treatment. Conclusion: Our findings reveal that NLRP3 inhibitor, OLT1177, protects against TMAO induced inflammation in endothelial cells and prevented tight junctional protein disruption safeguarding the endothelial barrier integrity. This work is supported by NIH R01 HL148711 award to Saisudha Koka This abstract was presented at the American Physiology Summit 2025 and is only available in HTML format. There is no downloadable file or PDF version. The Physiology editorial board was not involved in the peer review process.