title: Glutathione adducts on sarcoplasmic/endoplasmic reticulum Ca2+ ATPase Cys-674 regulate endothelial cell calcium stores and angiogenic function as well as promote ischemic blood flow recovery.
abstract: The sarco/endoplasmic reticulum Ca(2+) ATPase (SERCA) is key to Ca(2+) homeostasis and is redox-regulated by reversible glutathione (GSH) adducts on the cysteine (C) 674 thiol that stimulate Ca(2+) uptake activity and endothelial cell angiogenic responses in vitro. We found that mouse hind limb muscle ischemia induced S-glutathione adducts on SERCA in both whole muscle tissue and endothelial cells. To determine the role of S-glutathiolation, we used a SERCA 2 C674S heterozygote knock-in (SKI) mouse lacking half the key thiol. Following hind limb ischemia, SKI animals had decreased SERCA S-glutathione adducts and impaired blood flow recovery. We studied SKI microvascular endothelial cells in which total SERCA 2 expression was unchanged. Cultured SKI microvascular endothelial cells showed impaired migration and network formation compared with wild type (WT). Ca(2+) studies showed decreased nitric oxide (·NO)-induced (45)Ca(2+) uptake into the endoplasmic reticulum (ER) of SKI cells, while Fura-2 studies revealed lower Ca(2+) stores and decreased vascular endothelial growth factor (VEGF)- and ·NO-induced Ca(2+) influx. Adenoviral overexpression of calreticulin, an ER Ca(2+) binding protein, increased ionomycin-releasable stores, VEGF-induced Ca(2+) influx and endothelial cell migration. Taken together, these data indicate that the redox-sensitive Cys-674 thiol on SERCA 2 is required for normal endothelial cell Ca(2+) homeostasis and ischemia-induced angiogenic responses, revealing a novel redox control of angiogenesis via Ca(2+) stores.
authors.affiliation: From the Vascular Biology Section, Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Melissa D
authors.initials: MD
authors.lastname: Thompson
authors.affiliation: From the Vascular Biology Section, Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Yu
authors.initials: Y
authors.lastname: Mei
authors.affiliation: From the Vascular Biology Section, Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Robert M
authors.initials: RM
authors.lastname: Weisbrod
authors.affiliation: From the Vascular Biology Section, Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Marcy
authors.initials: M
authors.lastname: Silver
authors.affiliation: From the Vascular Biology Section, Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118.
authors.forename: Praphulla C
authors.initials: PC
authors.lastname: Shukla
authors.affiliation: the Ion Channel and Calcium Signaling Unit, Boston University School of Medicine, Boston, Massachusetts 02118, and.
authors.forename: Victoria M
authors.initials: VM
authors.lastname: Bolotina
authors.affiliation: From the Vascular Biology Section, Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118, racohen@bu.edu.
authors.forename: Richard A
authors.initials: RA
authors.lastname: Cohen
authors.affiliation: From the Vascular Biology Section, Department of Medicine, Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, Massachusetts 02118, the Innovative Drug Research Centre, Chongqing University, Chongqing 401331, China xy_tong@yahoo.com.
authors.forename: Xiaoyong
authors.initials: X
authors.lastname: Tong
mesh_terms: D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D042783:Endothelial Cells / Q000378:metabolism; D005260:Female; D055879:Gene Knock-In Techniques; D005978:Glutathione / Q000031:analogs & derivatives* / Q000378:metabolism; D006439:Hemodynamics; D006614:Hindlimb / Q000098:blood supply; D000860:Hypoxia / Q000201:enzymology / Q000503:physiopathology; D007511:Ischemia / Q000201:enzymology / Q000503:physiopathology; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D008817:Mice, Mutant Strains; D008822:Mice, Transgenic; D018482:Muscle, Skeletal / Q000098:blood supply / Q000201:enzymology; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018919:Neovascularization, Physiologic; D009569:Nitric Oxide / Q000378:metabolism; D010084:Oxidation-Reduction; D011247:Pregnancy; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D042461:Vascular Endothelial Growth Factor A; C467480:vascular endothelial growth factor A, mouse; D009569:Nitric Oxide; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; C506947:Atp2a2 protein, mouse; D005978:Glutathione; D002118:Calcium
keywords: Angiogenesis; Calcium; Calcium ATPase; Endothelial Cell; Hypoxia