NLRP3 Inflammasome Activation in Endothelial Cells and the Potential Modulatory Role of Riociguat in the Nitric Oxide Pathway

International Journal of Biomedicine 15(4) (2025) 752-755 http://dx.doi.org/10.21103/Article15(4)_OA18 ORIGINAL ARTICLE INTERNATIONAL JOURNAL OF BIOMEDICINE Experimental Biology NLRP3 Inflammasome Activation in Endothelial Cells and the Potential Modulatory Role of Riociguat in the Nitric Oxide Pathway Nita Kutllovci1, Alajdin Salihu2, Burim Neziri1,2* 1 2 Faculty of Medicine, University of Prishtina, Prishtina, Kosovo Institute of Pathophysiology, University Clinical Centre of Kosovo, Prishtina, Kosovo Abstract Background: The NOD-like receptor protein 3 (NLRP3) inflammasome is a cytoplasmic protein complex activated by damageassociated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), resulting in the release of proinflammatory cytokines such as IL-1β and IL-18. In this pathway, the activation of NF-κB plays a key role. Many studies have demonstrated the significant role of IL-1β in activating NF-κB. The aim of this study: We hypothesized that the activation of the nitric oxide (NO)- (sGC)-(cGMP)-PKG through the vasodilator-stimulated phosphoprotein (VASP) pathway may lead to antiinflammatory effects through (NFκB)-NLRP3 inhibition on endothelial cells. Methods and Results: This experimental research was conducted using human pulmonary artery endothelial cells (HPAEC). The growth and monitoring of cell cultures were done according to strict guidelines and protocols. In our study, we added TNFα (10 mg/mL) to activate the inflammasome. Ten minutes later, riociguat (RCG) was added at six different concentrations (50 µM, 10 µM, 5 µM, 1 µM, 0.5 µM, and 0.1 µM) to activate the NO pathway, followed by 15 mM of ATP. Incubation is continued at different times (60, 90, and 120 minutes). The measurement of caspase-1 activity was performed using a luminescence assay. Our results have once again demonstrated the successful activation of the inflammasome by TNFα. We suggest that the highest concentration of RCG, 50 µM, in our study was insufficient to trigger the NO pathway; additionally, more complex molecular pathways may be involved, and further investigations are warranted to clarify the underlying complex mechanism.(International Journal of Biomedicine. 2025;15(4):752-755.) Keywords: NLRP3 inflammasome • endothelial cells • nitric oxide • riociguat For citation: Kutllovci N, Salihu A, Neziri B. NLRP3 Inflammasome Activation in Endothelial Cells and the Potential Modulatory Role of Riociguat in the Nitric Oxide Pathway. International Journal of Biomedicine. 2025;15(4):752-755. doi:10.21103/ Article15(4)_OA18 Abbreviations DAMPs, damage-associated molecular patterns; HPAEC, human pulmonary artery endothelial cells; IL, interleukin; NLRP3, NOD-like receptor protein 3; NF-κB, nuclear factor-κB; PAMPs, and pathogen-associated molecular patterns; RCG, riociguat; VASP, the vasodilator-stimulated phosphoprotein. Introduction The NOD-like receptor protein 3 (NLRP3) inflammasome is a large and complex cytoplasmic protein that is activated when cells are exposed to pathogens or in response to cellular damage. The activation of caspase-1, a *Corresponding author: Burim Neziri, MD, PhD. Institute of Pathophysiology, University Clinical Centre of Kosovo, Prishtina, Kosovo E-mail: proteolytic enzyme, leads to the release of pro-inflammatory cytokines such as IL-1β and IL-18, thereby playing a crucial role in inflammation and tissue damage (Figure 1). Extended activation of the NLRP3 inflammasome may contribute to the development of chronic inflammatory diseases, including preeclampsia. 1-6 Within this scope, extensive research has been conducted on the significant levels of IL-1β in the activation of nuclear factor-κB (NF-κB), as observed in the pathogenesis of essential hypertension, pulmonary hypertension, gestational N. Kutllovci et al. / International Journal of Biomedicine 15(4) (2025) 752-755 hypertension, and preeclampsia. These studies have highlighted the important role of NF-κB in the inflammatory response and thrombosis.7-9 753 and cellular response via sGC stimulation. A possible molecular diagram describing the relationship between NO pathway activation and NLRP3 inflammasome activation was also reviewed to predict potential modulatory effects of new drugs. Methods Fig. 1. NLRP3 inflammasome role in the pathogenesis of inflammation and vasoconstriction Despite numerous publications suggesting that cyclic guanosine monophosphate (cGMP) inhibits NF-κB activation, the entire complex pathway, including the dynamics underlying this mechanism, remains incompletely understood. The NO–(cGMP)–(PDE-5) pathway is investigated in nearly all physiological, inflammatory, and neoplastic pathways. This molecular pathway is highly complex, and clarifying the role of any of its components would be greatly appreciated. This was further explained by recent publications, which have linked the phosphorylation of VASP (vasodilatorstimulated phosphoprotein) by cGMP-activated protein kinase G (PKG) to the NLRP3 inflammasome in hepatic cells.10 In this study, a connection between VASP phosphorylation status and NLRP3 activation, as well as the release of IL-β1 and IL-18, has been investigated. Since the role (cGMP) is now explained above, the role of its stimulants on the relaxation of smooth muscle cells in blood vessels is crucial (Figure 2). Therefore, we used riociguat (RCG), which directly stimulates soluble guanylyl cyclase (sGC), increasing so cGMP. This is particularly beneficial in cases where NO levels are decreased, such as in preeclampsia.11,12 Fig. 2. The cGMP-dependent pathway, initiated by nitric oxide (NO), is involved in vasodilation. Our study aimed to investigate the potential modulatory effects of RCG on endothelial cells through the NO pathway This experimental research was conducted using human pulmonary artery endothelial cells (HPAEC), which were thawed in a water bath. The special media were prepared with supplements, including fetal calf serum (0.02 ml/ ml), growth supplement for endothelial cells (0.004 ml/ml), epidermal growth factor (recombinant), fibroblast growth factor (recombinant), heparin, and hydrocortisone, as well as antibiotics (penicillin & streptomycin). Cells were seeded and incubated at 37°C with a carbon dioxide concentration of 5%. Medium changes were performed 24 hours after incubation and then every 2-3 days. After being confluent, about 70-90%, cells were detached with Trypsin/EDTA and centrifuged for 5 min at 1500. A laminin coating was performed using laminin at a concentration of 1mg/mL and incubated for 2 hours. Cells were cultured in 96-well plate culture and 6-well plate boxes, incubation followed. Cell treatment is as follows: initially, TNFα (10 µg/mL) is added, followed after 10 minutes by the addition of RCG in six concentrations (50 µM; 10 µM; 5 µM; 1 µM; 0.5 µM; 0.1 µM) and incubated for 12h at 37 °C with 5% CO2. Then, 15 mM of ATP is added, and the incubation is continued for 2 hours. Caspase- (...truncated)