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Endocytosed 2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils.

Okoshi T, Yamaguchi I, Ozawa D, Hasegawa K, Naiki H - PLoS ONE (2015)

Bottom Line: Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients.As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay.These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, Department of Pathological Sciences, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

ABSTRACT
Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.

No MeSH data available.


Related in: MedlinePlus

β2-m amyloid fibrils induce apoptosis of HIG-82 cells as measured by the TUNEL assay.After HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, TUNEL assay was performed as described in Materials and Methods. (A) The representative fluorescence images of TUNEL and DAPI double staining. The original magnification was x100. (B) The percentage of apoptotic cells to total cells. Data were presented as a dot plot of the ratios of five independent experiments with the mean value. Statistical analysis was performed by Mann-Whitney U-test. *P < 0.05.
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pone.0139330.g003: β2-m amyloid fibrils induce apoptosis of HIG-82 cells as measured by the TUNEL assay.After HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, TUNEL assay was performed as described in Materials and Methods. (A) The representative fluorescence images of TUNEL and DAPI double staining. The original magnification was x100. (B) The percentage of apoptotic cells to total cells. Data were presented as a dot plot of the ratios of five independent experiments with the mean value. Statistical analysis was performed by Mann-Whitney U-test. *P < 0.05.

Mentions: To further characterize the contribution of apoptosis to the cytotoxicity of β2-m amyloid fibrils, we performed the TUNEL assay as described in Materials and Methods (Fig 3). When HIG-82 cells were incubated with 100 μg/ml amyloid fibrils for 2 days, the percentage of apoptotic cells increased significantly as compared to β2-m monomer and vehicle buffer (2.9% of total cells vs. 0.3% and 0.3%, respectively; P<0.05 in both cases) (Fig 3B).


Endocytosed 2-Microglobulin Amyloid Fibrils Induce Necrosis and Apoptosis of Rabbit Synovial Fibroblasts by Disrupting Endosomal/Lysosomal Membranes: A Novel Mechanism on the Cytotoxicity of Amyloid Fibrils.

Okoshi T, Yamaguchi I, Ozawa D, Hasegawa K, Naiki H - PLoS ONE (2015)

β2-m amyloid fibrils induce apoptosis of HIG-82 cells as measured by the TUNEL assay.After HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, TUNEL assay was performed as described in Materials and Methods. (A) The representative fluorescence images of TUNEL and DAPI double staining. The original magnification was x100. (B) The percentage of apoptotic cells to total cells. Data were presented as a dot plot of the ratios of five independent experiments with the mean value. Statistical analysis was performed by Mann-Whitney U-test. *P < 0.05.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4589361&req=5

pone.0139330.g003: β2-m amyloid fibrils induce apoptosis of HIG-82 cells as measured by the TUNEL assay.After HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, TUNEL assay was performed as described in Materials and Methods. (A) The representative fluorescence images of TUNEL and DAPI double staining. The original magnification was x100. (B) The percentage of apoptotic cells to total cells. Data were presented as a dot plot of the ratios of five independent experiments with the mean value. Statistical analysis was performed by Mann-Whitney U-test. *P < 0.05.
Mentions: To further characterize the contribution of apoptosis to the cytotoxicity of β2-m amyloid fibrils, we performed the TUNEL assay as described in Materials and Methods (Fig 3). When HIG-82 cells were incubated with 100 μg/ml amyloid fibrils for 2 days, the percentage of apoptotic cells increased significantly as compared to β2-m monomer and vehicle buffer (2.9% of total cells vs. 0.3% and 0.3%, respectively; P<0.05 in both cases) (Fig 3B).

Bottom Line: Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients.As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay.These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Pathology, Department of Pathological Sciences, Faculty of Medical Sciences, University of Fukui, Fukui, Japan.

ABSTRACT
Dialysis-related amyloidosis is a major complication in long-term hemodialysis patients. In dialysis-related amyloidosis, β2-microglobulin (β2-m) amyloid fibrils deposit in the osteoarticular tissue, leading to carpal tunnel syndrome and destructive arthropathy with cystic bone lesions, but the mechanism by which these amyloid fibrils destruct bone and joint tissue is not fully understood. In this study, we assessed the cytotoxic effect of β2-m amyloid fibrils on the cultured rabbit synovial fibroblasts. Under light microscopy, the cells treated with amyloid fibrils exhibited both necrotic and apoptotic changes, while the cells treated with β2-m monomers and vehicle buffer exhibited no morphological changes. As compared to β2-m monomers and vehicle buffer, β2-m amyloid fibrils significantly reduced cellular viability as measured by the lactate dehydrogenase release assay and the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide reduction assay and significantly increased the percentage of apoptotic cells as measured by the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. β2-m amyloid fibrils added to the medium adhered to cell surfaces, but did not disrupt artificial plasma membranes as measured by the liposome dye release assay. Interestingly, when the cells were incubated with amyloid fibrils for several hours, many endosomes/lysosomes filled with amyloid fibrils were observed under confocal laser microscopy and electron microscopy, Moreover, some endosomal/lysosomal membranes were disrupted by intravesicular fibrils, leading to the leakage of the fibrils into the cytosol and adjacent to mitochondria. Inhibition of actin-dependent endocytosis by cytochalasin D attenuated the toxicity of amyloid fibrils. These results suggest that endocytosed β2-m amyloid fibrils induce necrosis and apoptosis by disrupting endosomal/lysosomal membranes, and this novel mechanism on the cytotoxicity of amyloid fibrils is described.

No MeSH data available.


Related in: MedlinePlus