Pyroptosis in Endothelial Cells and Extracellular Vesicle Release in Atherosclerosis via NF-κB-Caspase-4/5-GSDM-D Pathway
<b>Background</b>: Pyroptosis, an inflammatory cell death, is involved in the progression of atherosclerosis. Pyroptosis in endothelial cells (ECs) and its underlying mechanisms in atherosclerosis are poorly understood. Here, we investigated the role of a caspase-4/5-NF-κB pathway in pyr...
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2024-11-01
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| author | Salman Shamas Razia Rashid Rahil Laveena Kaushal Vinod Kumar Sharma Nissar Ahmad Wani Shabir H. Qureshi Sheikh F. Ahmad Sabry M. Attia Mohammad Afzal Zargar Abid Hamid Owais Mohmad Bhat |
| author_facet | Salman Shamas Razia Rashid Rahil Laveena Kaushal Vinod Kumar Sharma Nissar Ahmad Wani Shabir H. Qureshi Sheikh F. Ahmad Sabry M. Attia Mohammad Afzal Zargar Abid Hamid Owais Mohmad Bhat |
| author_sort | Salman Shamas |
| collection | DOAJ |
| description | <b>Background</b>: Pyroptosis, an inflammatory cell death, is involved in the progression of atherosclerosis. Pyroptosis in endothelial cells (ECs) and its underlying mechanisms in atherosclerosis are poorly understood. Here, we investigated the role of a caspase-4/5-NF-κB pathway in pyroptosis in palmitic acid (PA)-stimulated ECs and EVs as players in pyroptosis. <b>Methods</b>: Human umbilical vein endothelial cells (HUVECs) were cultured in an endothelial cell medium, treated with Ox-LDL, PA, caspase-4/5 inhibitor, NF-κB inhibitor, and sEV release inhibitor for 24 h, respectively. The cytotoxicity of PA was determined using an MTT assay, cell migration using a scratch-wound-healing assay, cell morphology using bright field microscopy, and lipid deposition using oil red O staining. The mRNA and protein expression of GSDM-D, CASP4, CASP5, NF-κB, NLRP3, IL-1β, and IL-18 were determined with RT-PCR and Western blot. Immunofluorescence was used to determine NLRP3 and ICAM-1 expressions. Extracellular vesicles (EVs) were isolated using an exosome isolation kit and were characterized by Western blot and scanning electron microscopy. <b>Results:</b> PA stimulation significantly changed the morphology of the HUVECs characterized by cell swelling, plasma membrane rupture, and increased LDH release, which are features of pyroptosis. PA significantly increased lipid accumulation and reduced cell migration. PA also triggered inflammation and endothelial dysfunction, as evidenced by NLRP3 activation, upregulation of ICAM-1 (endothelial activation marker), and pyroptotic markers (NLRP3, GSDM-D, IL-1β, IL-18). Inhibition of caspase-4/5 (Ac-FLTD-CMK) and NF-κB (trifluoroacetate salt (TFA)) resulted in a significant reduction in LDH release and expression of caspase-4/5, NF-κB, and gasdermin D (GSDM-D) in PA-treated HUVECs. Furthermore, GW4869, an exosome release inhibitor, markedly reduced LDH release in PA-stimulated HUVECs. EVs derived from PA-treated HUVECs exacerbated pyroptosis, as indicated by significantly increased LDH release and augmented expression of GSDM-D, NF-κB. <b>Conclusions:</b> The present study revealed that inflammatory, non-canonical caspase-4/5-NF-κB signaling may be one of the crucial mechanistic pathways associated with pyroptosis in ECs, and pyroptotic EVs facilitated pyroptosis in normal ECs during atherosclerosis. |
| format | Article |
| id | doaj-art-1aad42898094412582d7d654eb423292 |
| institution | Kabale University |
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| spelling | doaj-art-1aad42898094412582d7d654eb4232922024-12-27T14:45:43ZengMDPI AGPharmaceuticals1424-82472024-11-011712156810.3390/ph17121568Pyroptosis in Endothelial Cells and Extracellular Vesicle Release in Atherosclerosis via NF-κB-Caspase-4/5-GSDM-D PathwaySalman Shamas0Razia Rashid Rahil1Laveena Kaushal2Vinod Kumar Sharma3Nissar Ahmad Wani4Shabir H. Qureshi5Sheikh F. Ahmad6Sabry M. Attia7Mohammad Afzal Zargar8Abid Hamid9Owais Mohmad Bhat10Department of Biotechnology, School of Life Sciences, Central University of Kashmir, Ganderbal 191201, IndiaDepartment of Biotechnology, School of Life Sciences, Central University of Kashmir, Ganderbal 191201, IndiaDepartment of Dermatology, Venereology & Leprology, Postgraduate Institute for Medical Education and Research, Chandigarh 160012, IndiaDepartment of Dermatology, Venereology & Leprology, Postgraduate Institute for Medical Education and Research, Chandigarh 160012, IndiaDepartment of Biotechnology, School of Life Sciences, Central University of Kashmir, Ganderbal 191201, IndiaDepartment of Biotechnology, School of Life Sciences, Central University of Kashmir, Ganderbal 191201, IndiaDepartment of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi ArabiaDepartment of Pharmacology and Toxicology, College of Pharmacy, King Saud University, Riyadh 11451, Saudi ArabiaDepartment of Biotechnology, School of Life Sciences, Central University of Kashmir, Ganderbal 191201, IndiaDepartment of Biotechnology, School of Life Sciences, Central University of Kashmir, Ganderbal 191201, IndiaDepartment of Biotechnology, School of Life Sciences, Central University of Kashmir, Ganderbal 191201, India<b>Background</b>: Pyroptosis, an inflammatory cell death, is involved in the progression of atherosclerosis. Pyroptosis in endothelial cells (ECs) and its underlying mechanisms in atherosclerosis are poorly understood. Here, we investigated the role of a caspase-4/5-NF-κB pathway in pyroptosis in palmitic acid (PA)-stimulated ECs and EVs as players in pyroptosis. <b>Methods</b>: Human umbilical vein endothelial cells (HUVECs) were cultured in an endothelial cell medium, treated with Ox-LDL, PA, caspase-4/5 inhibitor, NF-κB inhibitor, and sEV release inhibitor for 24 h, respectively. The cytotoxicity of PA was determined using an MTT assay, cell migration using a scratch-wound-healing assay, cell morphology using bright field microscopy, and lipid deposition using oil red O staining. The mRNA and protein expression of GSDM-D, CASP4, CASP5, NF-κB, NLRP3, IL-1β, and IL-18 were determined with RT-PCR and Western blot. Immunofluorescence was used to determine NLRP3 and ICAM-1 expressions. Extracellular vesicles (EVs) were isolated using an exosome isolation kit and were characterized by Western blot and scanning electron microscopy. <b>Results:</b> PA stimulation significantly changed the morphology of the HUVECs characterized by cell swelling, plasma membrane rupture, and increased LDH release, which are features of pyroptosis. PA significantly increased lipid accumulation and reduced cell migration. PA also triggered inflammation and endothelial dysfunction, as evidenced by NLRP3 activation, upregulation of ICAM-1 (endothelial activation marker), and pyroptotic markers (NLRP3, GSDM-D, IL-1β, IL-18). Inhibition of caspase-4/5 (Ac-FLTD-CMK) and NF-κB (trifluoroacetate salt (TFA)) resulted in a significant reduction in LDH release and expression of caspase-4/5, NF-κB, and gasdermin D (GSDM-D) in PA-treated HUVECs. Furthermore, GW4869, an exosome release inhibitor, markedly reduced LDH release in PA-stimulated HUVECs. EVs derived from PA-treated HUVECs exacerbated pyroptosis, as indicated by significantly increased LDH release and augmented expression of GSDM-D, NF-κB. <b>Conclusions:</b> The present study revealed that inflammatory, non-canonical caspase-4/5-NF-κB signaling may be one of the crucial mechanistic pathways associated with pyroptosis in ECs, and pyroptotic EVs facilitated pyroptosis in normal ECs during atherosclerosis.https://www.mdpi.com/1424-8247/17/12/1568endothelial cellspyroptosisatherosclerosisinflammasomeendothelial dysfunctioninflammation |
| spellingShingle | Salman Shamas Razia Rashid Rahil Laveena Kaushal Vinod Kumar Sharma Nissar Ahmad Wani Shabir H. Qureshi Sheikh F. Ahmad Sabry M. Attia Mohammad Afzal Zargar Abid Hamid Owais Mohmad Bhat Pyroptosis in Endothelial Cells and Extracellular Vesicle Release in Atherosclerosis via NF-κB-Caspase-4/5-GSDM-D Pathway Pharmaceuticals endothelial cells pyroptosis atherosclerosis inflammasome endothelial dysfunction inflammation |
| title | Pyroptosis in Endothelial Cells and Extracellular Vesicle Release in Atherosclerosis via NF-κB-Caspase-4/5-GSDM-D Pathway |
| title_full | Pyroptosis in Endothelial Cells and Extracellular Vesicle Release in Atherosclerosis via NF-κB-Caspase-4/5-GSDM-D Pathway |
| title_fullStr | Pyroptosis in Endothelial Cells and Extracellular Vesicle Release in Atherosclerosis via NF-κB-Caspase-4/5-GSDM-D Pathway |
| title_full_unstemmed | Pyroptosis in Endothelial Cells and Extracellular Vesicle Release in Atherosclerosis via NF-κB-Caspase-4/5-GSDM-D Pathway |
| title_short | Pyroptosis in Endothelial Cells and Extracellular Vesicle Release in Atherosclerosis via NF-κB-Caspase-4/5-GSDM-D Pathway |
| title_sort | pyroptosis in endothelial cells and extracellular vesicle release in atherosclerosis via nf κb caspase 4 5 gsdm d pathway |
| topic | endothelial cells pyroptosis atherosclerosis inflammasome endothelial dysfunction inflammation |
| url | https://www.mdpi.com/1424-8247/17/12/1568 |
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