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Ca 2+ ‐dependent and Ceramide‐mediated Membrane Repair with Annexin V Recruitment and Aggregation in Mouse Endothelial Cells
Author(s) -
Chen Yang,
Xia Min,
Boini Krishna M.,
Li PinLan
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.944.10
Subject(s) - annexin , ceramide , acid sphingomyelinase , confocal microscopy , chemistry , microbiology and biotechnology , confocal , cell membrane , membrane , annexin a5 , cell , biology , biochemistry , apoptosis , geometry , mathematics
Annexin V is able to interact with biological membranes and may repair damaged cell membrane. It remains unknown whether this annexin V‐associated cell membrane repair can protect coronary endothelial cells (CECs) from a variety of injuries and whether is attributed to a Ca 2+ ‐dependent, ceramide‐mediated membrane repair in CECs. Using high energy Laser gun installed in confocal microscope, a tiny hole was made in CECs bathed with FM1‐43, and entry of FM1‐43 fluorescence was monitored to measure the extent of membrane injury. The cells exhibited a Ca 2+ dependent, ceramide‐mediated membrane repair, as shown by reduction of FM1‐43 fluorescence within CECs if they are bathed with a Ca 2+ ‐solution or pretreated by acid sphingomyelinase (ASM) inhibitor or siRNA. In CECs of ASM ‐/‐ mice, addition of ceramide also rescued the membrane repair response. By introduction of RFP‐annexin V into CECs, it was found that RFP‐annexin V were recruited and aggregated on the edge of a hole and accompanied with less FM1‐43 into cells. This ceramide‐mediated membrane repair was also studies in mouse streptozotocin (STZ) diabetic model. Ceramide and annexin V decreased in the coronary arterial endothelium by confocal and undetectable in ASM ‐/‐ coronary endothelium. Using YOYO‐1 and PI co‐staining with a time lag i.v. injection, confocal microscopic analysis of endothelial layer injury showed that more YOYO‐1 and PI staining on the arterial wall in STZ group, indicating less membrane resealing. These data show that Annexin V participated in a Ca 2+ ‐dependent and ceramide‐mediated membrane resealing process in CECs. In diabetes, this membrane resealing may be damaged leading to endothelial dysfunction, injury and ultimate sclerosis.