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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 adhere to the cell surfaces.HIG-82 cells incubated with Ham’s F12 medium containing 100 μg/ml β2-m fibrils (A-C) or vehicle buffer (D, E) for 6 hrs were stained with Congo red and observed with the confocal laser microscope as described in Materials and Methods. (B) and (E) are representative superimposed images on individual bright field micrographs. (C) A higher magnification of the box in (B). Images attached on the right and bottom are those of vertical sections on the lines intersecting at right angles. (A-C) When HIG-82 cells were incubated with 100 μg/ml amyloid fibrils for 6 hrs, they were firmly covered with amyloid fibrils. The scale bars are 50 μm long.
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pone.0139330.g004: β2-m amyloid fibrils adhere to the cell surfaces.HIG-82 cells incubated with Ham’s F12 medium containing 100 μg/ml β2-m fibrils (A-C) or vehicle buffer (D, E) for 6 hrs were stained with Congo red and observed with the confocal laser microscope as described in Materials and Methods. (B) and (E) are representative superimposed images on individual bright field micrographs. (C) A higher magnification of the box in (B). Images attached on the right and bottom are those of vertical sections on the lines intersecting at right angles. (A-C) When HIG-82 cells were incubated with 100 μg/ml amyloid fibrils for 6 hrs, they were firmly covered with amyloid fibrils. The scale bars are 50 μm long.

Mentions: To further characterize the effect of β2-m amyloid fibrils on HIG-82 cells, we performed Congo red staining and investigated whether β2-m amyloid fibrils directly interacted with the plasma membranes of HIG-82 cells. When HIG-82 cells were incubated with 100 μg/ml amyloid fibrils for 6 hrs, they were firmly covered with amyloid fibrils (Fig 4). This observation may indicate that β2-m amyloid fibrils adhered to the cell surface may directly injure the plasma membrane, leading to the release of LDH (Fig 2A). Thus, we next investigated the ability of β2-m amyloid fibrils to destruct artificial plasma membranes by the liposome dye release assay (Fig 5). As shown in Fig 5B and 5C, β2-m amyloid fibrils did not significantly destruct artificial plasma membranes of LUVs.


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 adhere to the cell surfaces.HIG-82 cells incubated with Ham’s F12 medium containing 100 μg/ml β2-m fibrils (A-C) or vehicle buffer (D, E) for 6 hrs were stained with Congo red and observed with the confocal laser microscope as described in Materials and Methods. (B) and (E) are representative superimposed images on individual bright field micrographs. (C) A higher magnification of the box in (B). Images attached on the right and bottom are those of vertical sections on the lines intersecting at right angles. (A-C) When HIG-82 cells were incubated with 100 μg/ml amyloid fibrils for 6 hrs, they were firmly covered with amyloid fibrils. The scale bars are 50 μm long.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0139330.g004: β2-m amyloid fibrils adhere to the cell surfaces.HIG-82 cells incubated with Ham’s F12 medium containing 100 μg/ml β2-m fibrils (A-C) or vehicle buffer (D, E) for 6 hrs were stained with Congo red and observed with the confocal laser microscope as described in Materials and Methods. (B) and (E) are representative superimposed images on individual bright field micrographs. (C) A higher magnification of the box in (B). Images attached on the right and bottom are those of vertical sections on the lines intersecting at right angles. (A-C) When HIG-82 cells were incubated with 100 μg/ml amyloid fibrils for 6 hrs, they were firmly covered with amyloid fibrils. The scale bars are 50 μm long.
Mentions: To further characterize the effect of β2-m amyloid fibrils on HIG-82 cells, we performed Congo red staining and investigated whether β2-m amyloid fibrils directly interacted with the plasma membranes of HIG-82 cells. When HIG-82 cells were incubated with 100 μg/ml amyloid fibrils for 6 hrs, they were firmly covered with amyloid fibrils (Fig 4). This observation may indicate that β2-m amyloid fibrils adhered to the cell surface may directly injure the plasma membrane, leading to the release of LDH (Fig 2A). Thus, we next investigated the ability of β2-m amyloid fibrils to destruct artificial plasma membranes by the liposome dye release assay (Fig 5). As shown in Fig 5B and 5C, β2-m amyloid fibrils did not significantly destruct artificial plasma membranes of LUVs.

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