Mapping mechanisms and charting the time course of premature cell senescence and apoptosis: lysosomal dysfunction and ganglioside accumulation in endothelial cells.
Endothelial cells subjected to glycated collagen I develop premature senescence within 3-5 days, as revealed by increased senescence-associated beta-galactosidase activity, decreased proliferation, and an increase in cell size. Here, we analyzed the time course and possible mechanisms of this process. Lysosomal integrity studies revealed a rapid collapse of pH gradient and lysosomal permeabilization, detectable after 30 min, and preceded by the increased production of reactive oxygen species. Measurement of mitochondrial membrane potential after application of glycated collagen demonstrated that depolarization was delayed by 4 h compared with changes in lysosomal pH and permeability. Based on the above findings of lysosomal permeabilization, we hypothesized that the reduced activity of senescence-associated beta-galactosidase could be responsible for the cellular accumulation of gangliosides, previously shown to induce cell senescence. After 5 days of exposure to glycated collagen, there was an increase in the levels of gangliosides GM3, GD1b, and GT1b, coincident with development of cell senescence. Treatment of endothelial cells with d-threo-EtDOP4, an inhibitor of glucosylceramide synthase, inhibited apoptosis, but not the development of senescence. In conclusion, collagen I modified by advanced glycation initially induces apoptosis of human umbilical vein endothelial cells. This process is initiated by the collapse of lysosomal pH and an increase in lysosomal permeability, with the subsequent mitochondrial depolarization and accumulation of gangliosides. Blockade of ganglioside synthesis suppresses apoptosis, but not senescence, which develops after 3 days of exposure to glycated collagen. These data imply a critical role for lysosomal permeabilization in triggering apoptosis of endothelial cells exposed to the diabetic milieu.
Patschan
Susann
S
Department of Medicine, New York Medical College, Valhalla, New York, USA. spatschan@gmail.comor
Chen
Jun
J
Gealekman
Olga
O
Krupincza
Katrina
K
Wang
Maureen
M
Shu
Liming
L
Shayman
James A
JA
Goligorsky
Michael S
MS
D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D002460:Cell Line; D002478:Cells, Cultured; D016922:Cellular Senescence / Q000502:physiology*; D024042:Collagen Type I / Q000494:pharmacology; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D005732:Gangliosides / Q000378:metabolism*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008247:Lysosomes / Q000187:drug effects / Q000502:physiology*; D008564:Membrane Potentials / Q000187:drug effects / Q000502:physiology; D051379:Mice; D013997:Time Factors
D024042:Collagen Type I; D005732:Gangliosides