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

ORIGINAL ARTICLE
Nita Kutllovci, Alajdin Salihu, Burim Neziri

 
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
 
Originally published December 5, 2025

Abstract: 

Background: The NOD-like receptor protein 3 (NLRP3) inflammasome is a cytoplasmic protein complex activated by damage-associated molecular patterns (DAMPs) and pathogen-associated molecular patterns (PAMPs), resulting in the release of pro-inflammatory 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 anti-inflammatory 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.

Keywords: 
NLRP3 inflammasome • endothelial cells • nitric oxide • riociguat
References: 
  1. Scott TE, Kemp-Harper BK, Hobbs AJ. Inflammasomes: a novel therapeutic target in pulmonary hypertension? Br J Pharmacol. 2019 Jun;176(12):1880-1896. doi: 10.1111/bph.14375. Epub 2018 Jun 27. PMID: 29847700; PMCID: PMC6534811.
  2. Latz E, Xiao TS, Stutz A. Activation and regulation of the inflammasomes. Nat Rev Immunol. 2013 Jun;13(6):397-411. doi: 10.1038/nri3452. PMID: 23702978; PMCID: PMC3807999.
  3. Swanson KV, Deng M, Ting JP. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol. 2019 Aug;19(8):477-489. doi: 10.1038/s41577-019-0165-0. PMID: 31036962; PMCID: PMC7807242.
  4. Zhou F, Li C, Zhang SY. NLRP3 inflammasome: a new therapeutic target for high-risk reproductive disorders? Chin Med J (Engl). 2020 Nov 4;134(1):20-27. doi: 10.1097/CM9.0000000000001214. PMID: 33395071; PMCID: PMC7862815.
  5. Shirasuna K, Karasawa T, Takahashi M. Role of the NLRP3 Inflammasome in Preeclampsia. Front Endocrinol (Lausanne). 2020 Feb 25;11:80. doi: 10.3389/fendo.2020.00080. PMID: 32161574; PMCID: PMC7053284.
  6. Pasqua T, Pagliaro P, Rocca C, Angelone T, Penna C. Role of NLRP-3 Inflammasome in Hypertension: A Potential Therapeutic Target. Curr Pharm Biotechnol. 2018;19(9):708-714. doi: 10.2174/1389201019666180808162011. PMID: 30091406.
  7. Lawrence T. The nuclear factor NF-kappaB pathway in inflammation. Cold Spring Harb Perspect Biol. 2009 Dec;1(6):a001651. doi: 10.1101/cshperspect.a001651. Epub 2009 Oct 7. PMID: 20457564; PMCID: PMC2882124.
  8. Vaughan JE, Walsh SW. Activation of NF-κB in placentas of women with preeclampsia. Hypertens Pregnancy. 2012;31(2):243-51. doi: 10.3109/10641955.2011.642436. Epub 2012 Mar 1. PMID: 22380486; PMCID: PMC3542769.
  9. Mussbacher M, Salzmann M, Brostjan C, Hoesel B, Schoergenhofer C, Datler H, Hohensinner P, Basílio J, Petzelbauer P, Assinger A, Schmid JA. Cell Type-Specific Roles of NF-κB Linking Inflammation and Thrombosis. Front Immunol. 2019 Feb 4;10:85. doi: 10.3389/fimmu.2019.00085. PMID: 30778349; PMCID: PMC6369217.
  10. Laban H, Weigert A, Zink J, Elgheznawy A, Schürmann C, Günther L, Abdel Malik R, Bothur S, Wingert S, Bremer R, Rieger MA, Brüne B, Brandes RP, Fleming I, Benz PM. VASP regulates leukocyte infiltration, polarization, and vascular repair after ischemia. J Cell Biol. 2018 Apr 2;217(4):1503-1519. doi: 10.1083/jcb.201702048. Epub 2018 Mar 5. PMID: 29507126; PMCID: PMC5881493.
  11. Benza R, Mathai S, Nathan SD. sGC stimulators: Evidence for riociguat beyond groups 1 and 4 pulmonary hypertension. Respir Med. 2017 Jan;122 Suppl 1:S28-S34. doi: 10.1016/j.rmed.2016.11.010. Epub 2016 Nov 14. PMID: 27890470.
  12. Ghofrani HA, Galiè N, Grimminger F, Grünig E, Humbert M, Jing ZC, Keogh AM, Langleben D, Kilama MO, Fritsch A, Neuser D, Rubin LJ; PATENT-1 Study Group. Riociguat for the treatment of pulmonary arterial hypertension. N Engl J Med. 2013 Jul 25;369(4):330-40. doi: 10.1056/NEJMoa1209655. PMID: 23883378.
  13. Kutllovci N. Investigating the correlation between activation of the Nitric Oxide pathway and inhibition of the NLRP3 inflammasome in endothelial cells in preeclampsia. Italian Journal JOC. 2024;36 (Supplement No. 2):25. doi: 10.36129/jog.2024.S63
  14. Kahlenberg JM, Lundberg KC, Kertesy SB, Qu Y, Dubyak GR. Potentiation of caspase-1 activation by the P2X7 receptor is dependent on TLR signals and requires NF-kappaB-driven protein synthesis. J Immunol. 2005 Dec 1;175(11):7611-22. doi: 10.4049/jimmunol.175.11.7611. PMID: 16301671.
  15. Usui F, Shirasuna K, Kimura H, Tatsumi K, Kawashima A, Karasawa T, Hida S, Sagara J, Taniguchi S, Takahashi M. Critical role of caspase-1 in vascular inflammation and development of atherosclerosis in Western diet-fed apolipoprotein E-deficient mice. Biochem Biophys Res Commun. 2012 Aug 24;425(2):162-8. doi: 10.1016/j.bbrc.2012.07.058. Epub 2012 Jul 20. PMID: 22819845.
  16. Lopez-Pastrana J, Ferrer LM, Li YF, Xiong X, Xi H, Cueto R, Nelson J, Sha X, Li X, Cannella AL, Imoukhuede PI, Qin X, Choi ET, Wang H, Yang XF. Inhibition of Caspase-1 Activation in Endothelial Cells Improves Angiogenesis: A NOVEL THERAPEUTIC POTENTIAL FOR ISCHEMIA. J Biol Chem. 2015 Jul 10;290(28):17485-94. doi: 10.1074/jbc.M115.641191. Epub 2015 Jun 2. PMID: 26037927; PMCID: PMC4498083.
  17. Melton E, Qiu H. Interleukin-1β in Multifactorial Hypertension: Inflammation, Vascular Smooth Muscle Cell and Extracellular Matrix Remodeling, and Non-Coding RNA Regulation. Int J Mol Sci. 2021 Aug 11;22(16):8639. doi: 10.3390/ijms22168639. PMID: 34445357; PMCID: PMC8395428.
  18. Kim JH, Kim JY, Park M, Kim S, Kim T, Kim J, Choi S, Park W, Hwang JY, Choe J, Ha KS, Won MH, Ryoo S, Kwon YG, Kim YM. NF-κB-dependent miR-31/155 biogenesis is essential for TNF-α-induced impairment of endothelial progenitor cell function. Exp Mol Med. 2020 Aug;52(8):1298-1309. doi: 10.1038/s12276-020-0478-x. Epub 2020 Aug 7. PMID: 32770080; PMCID: PMC8080610.
  19. Gao Q, Tang J, Li N, Zhou X, Li Y, Liu Y, Wu J, Yang Y, Shi R, He A, Li X, Zhang Y, Chen J, Zhang L, Sun M, Xu Z. A novel mechanism of angiotensin II-regulated placental vascular tone in the development of hypertension in preeclampsia. Oncotarget. 2017 May 9;8(19):30734-30741. doi: 10.18632/oncotarget.15416. PMID: 28430615; PMCID: PMC5458163.
  20. Zhou R, Yazdi AS, Menu P, Tschopp J. A role for mitochondria in NLRP3 inflammasome activation. Nature. 2011 Jan 13;469(7329):221-5. doi: 10.1038/nature09663. Epub 2010 Dec 1. Erratum in: Nature. 2011 Jul 7;475(7354):122. PMID: 21124315.
  21. Tanase DM, Valasciuc E, Gosav EM, Ouatu A, Buliga-Finis ON, Floria M, Maranduca MA, Serban IL. Portrayal of NLRP3 Inflammasome in Atherosclerosis: Current Knowledge and Therapeutic Targets. Int J Mol Sci. 2023 May 3;24(9):8162. doi: 10.3390/ijms24098162. PMID: 37175869; PMCID: PMC10179095.
  22. Benz PM, Frömel T, Laban H, Zink J, Ulrich L, Groneberg D, Boon RA, Poley P, Renne T, de Wit C, Fleming I. Cardiovascular Functions of Ena/VASP Proteins: Past, Present and Beyond. Cells. 2023 Jun 28;12(13):1740. doi: 10.3390/cells12131740. PMID: 37443774; PMCID: PMC10340426.

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Received August 16, 2025.
Accepted September 6, 2025.
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