| 1. |
- Bankell, Elisabeth, et al.
(författare)
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LL-37-induced caspase-independent apoptosis is associated with plasma membrane permeabilization in human osteoblast-like cells
- 2021
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Ingår i: Peptides. - : Elsevier BV. - 0196-9781 .- 1873-5169. ; 135
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Tidskriftsartikel (refereegranskat)abstract
- The host defense peptide LL-37 is active against both gram-positive and gram-negative bacteria, but it has also been shown to reduce human host cell viability. However, the mechanisms behind LL-37-induced human host cell cytotoxicity are not yet fully understood. Here, we assess if LL-37-evoked attenuation of human osteoblast-like MG63 cell viability is associated with apoptosis, and if the underlying mechanism may involve LL-37-induced plasma membrane permeabilization. MG63 cell viability and plasma membrane permeabilization were investigated by using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and by measuring lactate dehydrogenase (LDH) release, respectively. Apoptosis was assessed by the terminal deoxynucleotidyl dUTP nick end labeling (TUNEL) assay and Annexin V flow cytometry, and caspase-3 and poly (ADP-ribose) polymerase (PARP) cleavage were determined by Western blot. LL-37 (4 and 10 μM) reduced both cell number and cell viability, and these effects were associated with a pro-apoptotic effect demonstrated by positive TUNEL staining and Annexin V flow cytometry. LL-37-induced apoptosis was not coupled to either caspase-3 or PARP cleavage, suggesting that LL-37 causes caspase-independent apoptosis in MG63 cells. Both LL-37 and the well-known plasma membrane permeabilizer Triton X-100 reduced cell viability and stimulated LDH release. Triton X-100-treated cells showed positive TUNEL staining, and the detergent accumulated cells in late apoptosis/necrosis. Similar to LL-37, Triton X-100 caused no PARP cleavage. We conclude that LL-37 promotes caspase-independent apoptosis, and that this effect seems coupled to plasma membrane permeabilization in human MG63 cells.
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| 2. |
- Dahl, Sara, et al.
(författare)
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The host defense peptide LL-37 triggers release of nucleic acids from human mast cells
- 2018
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Ingår i: Peptides. - : Elsevier BV. - 0196-9781 .- 1873-5169. ; 109, s. 39-45
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Tidskriftsartikel (refereegranskat)abstract
- The human host defense peptide LL-37 possesses antimicrobial activity but also affects host cell function and viability. Mast cells are involved in innate immunity but no data have been presented on effects of LL-37 on human mast cell viability and export of nucleic acids. Here, we demonstrated by immunofluorescence microscopy that synthesized LL-37 was internalized by human LAD2 mast cells and detected both in cytoplasm and nucleus. Treatment with high (4 and 10 μM) but not low (1 μM) concentrations of LL-37 for 4 h reduced cell viability assessed by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Stimulation with 10 μM LL-37 for 4 h enhanced export of nucleic acids, total protein and lactate dehydrogenase (LDH), suggesting that both nuclear and plasma membranes are permeabilized by LL-37. Although LL-37 triggered release of nucleic acids, no extracellular trap-like structures were observed by laser scanning confocal microscopy of cells incubated with the plasma membrane impermeable nucleic acid fluorophore SYTOX-Green, indicating that LL-37 promotes export of nucleic acids but not formation of extracellular traps. On the other hand, phorbol-12-myristate-13-acetate (PMA), which is a well-known inducer of extracellular traps, stimulated export of nucleic acids and also formation of extracellular trap-like structures. However, PMA had no effect on export of either total protein or LDH. Hence, LL-37 and PMA seem to stimulate export of nucleic acids from LAD2 mast cells through different pathways. In conclusion, we demonstrate that LL-37 triggers release of nucleic acids from human mast cells but not the formation of extracellular trap-like structures.
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| 3. |
- Svensson, Daniel, et al.
(författare)
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Inhibition of MicroRNA-125a Promotes Human Endothelial Cell Proliferation and Viability through an Antiapoptotic Mechanism.
- 2014
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Ingår i: Journal of Vascular Research. - : S. Karger AG. - 1423-0135 .- 1018-1172. ; 51:3, s. 239-245
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Tidskriftsartikel (refereegranskat)abstract
- The microRNA-125a (miR-125a) is highly expressed in endothelial cells, but its role in vascular biology is not known. Endothelial cell proliferation and viability play an important role in endothelial healing, and we hypothesize that miR-125a regulates this process. The aim of the present study was to investigate if miR-125a controls human endothelial cell proliferation, viability and endothelial healing, and to assess the mechanisms involved. We showed that overexpression of miR-125a by transfection with miR-125a mimic reduced human umbilical vein endothelial cell (HUVEC) proliferation and viability, and stimulated apoptosis as demonstrated by a miR-125a-induced increase of the proportion of annexin V-positive cells monitored by flow cytometry. Moreover, we showed that the miR-125a mimic downregulated the antiapoptotic Bcl2 protein and upregulated caspase 3, suggesting that these two proteins represent molecular targets for miR-125a. Accordingly, transfection with miR-125a inhibitor, downregulating miR-125a expression, promoted HUVEC proliferation and viability, and reduced apoptosis. Importantly, transfection with miR-125a inhibitor promoted HUVEC tube formation in Matrigel, suggesting that reduction of miR-125a has a proangiogenic effect. In conclusion, downregulation of miR-125a through local transfection with miR-125a inhibitor might be a new way to enhance endothelial cell proliferation and viability, thereby promoting the reendothelialization observed in response to intimal injury. © 2014 S. Karger AG, Basel.
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| 4. |
- Säll, Johanna, et al.
(författare)
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The antimicrobial peptide LL-37 alters human osteoblast Ca2+ handling and induces Ca2+-independent apoptosis
- 2013
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Ingår i: Journal of Innate Immunity. - : S. Karger. - 1662-811X .- 1662-8128. ; 3:5, s. 290-300
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Tidskriftsartikel (refereegranskat)abstract
- The human antimicrobial peptide cathelicidin LL-37 has, besides its antimicrobial properties, also been shown to regulate apoptosis in a cell type-specific manner. Mechanisms involved in LL-37-regulated apoptotic signaling are not identified. Here, we show that LL-37 reduces the human osteoblast-like MG63 cell number and cell viability in the micromolar concentration range with an IC50 value of about 5 µM. Treatment with 4 µM LL-37 increased the number of annexin V-positive cells and stimulated activation of caspase 3 showing that LL-37 promotes apoptosis. Treatment with 4 µM LL-37 caused an acute and sustained rise in intracellular Ca(2+) concentration assessed by laser-scanning confocal microscopy of Fluo-4-AM-loaded MG63 cells. LL-37 increased Ca(2+) also in the presence of the respective L- and T-type voltage-sensitive Ca(2+) channel blockers nifedipine and NiCl2. LL-37 had no effect on Ca(2+) in cells incubated with Ca(2+)-free solution. LL-37 (4 and 8 µM) reduced the MG63 cell number both in the presence and absence of Ca(2+) in the medium. In conclusion, LL-37 reduces the osteoblast cell number by promoting apoptosis, and furthermore, LL-37 stimulates Ca(2+) inflow via a mechanism independent of voltage-sensitive Ca(2+) channels. Interestingly, LL-37-induced lowering of the cell number seems to be mediated via a mechanism independent of Ca(2+).
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