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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 reduce cellular viability of HIG-82 cells.After HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer, 10 or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, LDH releasing assay (A) and MTT reduction assay (B) were performed as described in Materials and Methods. Data normalized to positive control and vehicle in LDH releasing assay and MTT reduction assay, respectively were presented as mean ± SD of three independent experiments. Statistical analysis was performed by Student’s unpaired t-test. *P < 0.05, **P < 0.001, #P < 0.0001.
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pone.0139330.g002: β2-m amyloid fibrils reduce cellular viability of HIG-82 cells.After HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer, 10 or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, LDH releasing assay (A) and MTT reduction assay (B) were performed as described in Materials and Methods. Data normalized to positive control and vehicle in LDH releasing assay and MTT reduction assay, respectively were presented as mean ± SD of three independent experiments. Statistical analysis was performed by Student’s unpaired t-test. *P < 0.05, **P < 0.001, #P < 0.0001.

Mentions: We next quantified the cytotoxic effect of β2-m amyloid fibrils on HIG-82 cells by LDH releasing assay and MTT reduction assay as described in Materials and Methods. In LDH releasing assay (Fig 2A), 100 μg/ml β2-m amyloid fibrils reduced cellular viability significantly as compared to β2-m monomer and vehicle buffer (54.2 ± 5.1% of positive control vs. 11.2 ± 0.6% and 11.7 ± 0.5%, respectively; P<0.0001 in both cases). 10 μg/ml β2-m amyloid fibrils exhibited no significant cytotoxicity. These data suggest that β2-m amyloid fibrils induced the necrosis of HIG-82 cells, leading to the rupture of plasma membranes [47, 48]. In the MTT reduction assay (Fig 2B), 100 μg/ml β2-m amyloid fibrils reduced cellular viability significantly as compared to β2-m monomer and vehicle (25.3 ± 0.3% of vehicle vs. 93.3 ± 5.5% and 100%, respectively; P<0.0001 in both cases). 10 μg/ml β2-m amyloid fibrils also exhibited significant cytotoxicity as compared to vehicle (77.0 ± 12.5% of vehicle; P<0.05). The supernatant of the fibril preparation added to the cells did not affect cellular viability of HIG-82 cells as measured by the LDH releasing assay and the MTT reduction assay (S4 Fig), indicating that residual SDS contamination is not responsible for the observed cytotoxicity.


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 reduce cellular viability of HIG-82 cells.After HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer, 10 or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, LDH releasing assay (A) and MTT reduction assay (B) were performed as described in Materials and Methods. Data normalized to positive control and vehicle in LDH releasing assay and MTT reduction assay, respectively were presented as mean ± SD of three independent experiments. Statistical analysis was performed by Student’s unpaired t-test. *P < 0.05, **P < 0.001, #P < 0.0001.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139330.g002: β2-m amyloid fibrils reduce cellular viability of HIG-82 cells.After HIG-82 cells were incubated with Ham’s F12 medium containing vehicle buffer, 10 or 100 μg/ml β2-m fibrils or r-β2-m monomer for 2 days, LDH releasing assay (A) and MTT reduction assay (B) were performed as described in Materials and Methods. Data normalized to positive control and vehicle in LDH releasing assay and MTT reduction assay, respectively were presented as mean ± SD of three independent experiments. Statistical analysis was performed by Student’s unpaired t-test. *P < 0.05, **P < 0.001, #P < 0.0001.
Mentions: We next quantified the cytotoxic effect of β2-m amyloid fibrils on HIG-82 cells by LDH releasing assay and MTT reduction assay as described in Materials and Methods. In LDH releasing assay (Fig 2A), 100 μg/ml β2-m amyloid fibrils reduced cellular viability significantly as compared to β2-m monomer and vehicle buffer (54.2 ± 5.1% of positive control vs. 11.2 ± 0.6% and 11.7 ± 0.5%, respectively; P<0.0001 in both cases). 10 μg/ml β2-m amyloid fibrils exhibited no significant cytotoxicity. These data suggest that β2-m amyloid fibrils induced the necrosis of HIG-82 cells, leading to the rupture of plasma membranes [47, 48]. In the MTT reduction assay (Fig 2B), 100 μg/ml β2-m amyloid fibrils reduced cellular viability significantly as compared to β2-m monomer and vehicle (25.3 ± 0.3% of vehicle vs. 93.3 ± 5.5% and 100%, respectively; P<0.0001 in both cases). 10 μg/ml β2-m amyloid fibrils also exhibited significant cytotoxicity as compared to vehicle (77.0 ± 12.5% of vehicle; P<0.05). The supernatant of the fibril preparation added to the cells did not affect cellular viability of HIG-82 cells as measured by the LDH releasing assay and the MTT reduction assay (S4 Fig), indicating that residual SDS contamination is not responsible for the observed cytotoxicity.

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