Advanced glycation end-products attenuate human mesenchymal stem cells and prevent cognate differentiation into adipose tissue, cartilage, and bone.
The impact of AGEs on human MSCs was studied. AGEs inhibited the proliferation of MSCs, induced apoptosis, and prevented cognate differentiation into adipose tissue, cartilage, and bone, suggesting a deleterious effect of AGEs in the pathogenesis of musculoskeletal disorders in aged and diabetic patients.


BACKGROUND
Advanced glycation end-products (AGEs) are accumulated on long-lived proteins of various tissues in advanced age and diabetes mellitus and have been implicated in chronic complication, including musculoskeletal disorders. Human mesenchymal stem cells (MSCs) potentially differentiate into mature musculoskeletal tissues during tissue repair, but the pathogenetic role of AGEs on MSCs is unclear.


METHODS
AGEs were prepared by incubating BSA with glucose, glyceraldehydes, or glycolaldehyde (designated as AGE-1, AGE-2, or AGE-3, respectively). Proliferation, apoptosis, and reactive oxygen species (ROS) generation were assayed in AGE-treated cells. The expression of the receptor for AGE (RAGE) was examined by immunohistochemistry and Western blotting. Involvement of RAGE-mediated signaling was examined using a neutralizing antiserum against RAGE. Differentiation into adipose tissue, cartilage, and bone were morphologically and biochemically monitored with specific markers for each.


RESULTS
AGE-2 and AGE-3, but not control nonglycated BSA and AGE-1, reduced the viable cell number and 5-bromo-2'deoxyuridine (BrdU) incorporation with increased intracellular ROS generation and the percentage of apoptotic cells. MSCs expressed RAGE and its induction was stimulated by AGE-2 and AGE-3. These AGEs inhibited adipogenic differentiation (assayed by oil red O staining, lipoprotein lipase production, and intracellular triglyceride content) and chondrogenic differentiation (assayed by safranin O staining and type II collagen production). On osteogenic differentiation, AGE-2 and AGE-3 increased alkaline phosphatase activity and intracellular calcium content; however, von Kossa staining revealed the loss of mineralization and mature bone nodule formation. The antiserum against RAGE partially prevented AGE-induced cellular events.


CONCLUSIONS
AGE-2 and AGE-3 may lead to the in vivo loss of MSC mass and the delay of tissue repair by inhibiting the maturation of MSC-derived cells. The AGE-RAGE interaction may be involved in the deleterious effect of AGEs on MSCs.
Kume
Shinichiro
S
Department of Orthopaedic Surgery, Kurume University School of Medicine, Kurume, Japan.
Kato
Seiya
S
Yamagishi
Sho-ichi
S
Inagaki
Yosuke
Y
Ueda
Seiji
S
Arima
Nobuyuki
N
Okawa
Takahiro
T
Kojiro
Masamichi
M
Nagata
Kensei
K
D000079:Acetaldehyde / Q000031:analogs & derivatives / Q000494:pharmacology; D017667:Adipocytes / Q000166:cytology; D000273:Adipose Tissue / Q000166:cytology / Q000473:pathology*; D017209:Apoptosis; D015153:Blotting, Western; D001842:Bone and Bones / Q000166:cytology / Q000473:pathology*; D001973:Bromodeoxyuridine / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002356:Cartilage / Q000166:cytology / Q000473:pathology*; D002454:Cell Differentiation / Q000187:drug effects*; D002460:Cell Line; D049109:Cell Proliferation; D002470:Cell Survival; D002478:Cells, Cultured; D019902:Chondrocytes / Q000166:cytology; D024043:Collagen Type II / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay; D005947:Glucose / Q000494:pharmacology; D017127:Glycation End Products, Advanced / Q000378:metabolism / Q000502:physiology*; D005985:Glyceraldehyde / Q000494:pharmacology; D006801:Humans; D007150:Immunohistochemistry; D008071:Lipoprotein Lipase / Q000096:biosynthesis; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D008856:Microscopy, Fluorescence; D016253:Microscopy, Immunoelectron; D008858:Microscopy, Phase-Contrast; D010011:Osteocytes / Q000378:metabolism; D010619:Phenazines / Q000494:pharmacology; D017382:Reactive Oxygen Species; D013234:Stem Cells / Q000166:cytology; D013997:Time Factors; D014945:Wound Healing
D024043:Collagen Type II; D017127:Glycation End Products, Advanced; D010619:Phenazines; D017382:Reactive Oxygen Species; D005985:Glyceraldehyde; D008071:Lipoprotein Lipase; D001973:Bromodeoxyuridine; D000079:Acetaldehyde; D005947:Glucose; D002118:Calcium; C010972:glycolaldehyde; C009195:safranine T