title: Restoration of autophagy in endothelial cells from patients with diabetes mellitus improves nitric oxide signaling.
abstract: Endothelial dysfunction contributes to cardiovascular disease in diabetes mellitus. Autophagy is a multistep mechanism for the removal of damaged proteins and organelles from the cell. Under diabetic conditions, inadequate autophagy promotes cellular dysfunction and insulin resistance in non-vascular tissue. We hypothesized that impaired autophagy contributes to endothelial dysfunction in diabetes mellitus.
  
  
  
  We measured autophagy markers and endothelial nitric oxide synthase (eNOS) activation in freshly isolated endothelial cells from diabetic subjects (n = 45) and non-diabetic controls (n = 41). p62 levels were higher in cells from diabetics (34.2 ± 3.6 vs. 20.0 ± 1.6, P = 0.001), indicating reduced autophagic flux. Bafilomycin inhibited insulin-induced activation of eNOS (64.7 ± 22% to -47.8 ± 8%, P = 0.04) in cells from controls, confirming that intact autophagy is necessary for eNOS signaling. In endothelial cells from diabetics, activation of autophagy with spermidine restored eNOS activation, suggesting that impaired autophagy contributes to endothelial dysfunction (P = 0.01). Indicators of autophagy initiation including the number of LC3-bound puncta and beclin 1 expression were similar in diabetics and controls, whereas an autophagy terminal phase indicator, the lysosomal protein Lamp2a, was higher in diabetics. In endothelial cells under diabetic conditions, the beneficial effect of spermidine on eNOS activation was blocked by autophagy inhibitors bafilomycin or 3-methyladenine. Blocking the terminal stage of autophagy with bafilomycin increased p62 (P = 0.01) in cells from diabetics to a lesser extent than in cells from controls (P = 0.04), suggesting ongoing, but inadequate autophagic clearance.
  
  
  
  Inadequate autophagy contributes to endothelial dysfunction in patients with diabetes and may be a target for therapy of diabetic vascular disease.
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Jessica L
authors.initials: JL
authors.lastname: Fetterman
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Monica
authors.initials: M
authors.lastname: Holbrook
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Nir
authors.initials: N
authors.lastname: Flint
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Bihua
authors.initials: B
authors.lastname: Feng
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Rosa
authors.initials: R
authors.lastname: Bretón-Romero
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Erika A
authors.initials: EA
authors.lastname: Linder
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Brittany D
authors.initials: BD
authors.lastname: Berk
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Mai-Ann
authors.initials: MA
authors.lastname: Duess
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Melissa G
authors.initials: MG
authors.lastname: Farb
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Noyan
authors.initials: N
authors.lastname: Gokce
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Orian S
authors.initials: OS
authors.lastname: Shirihai
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA. Electronic address: nhamburg@bu.edu.
authors.forename: Naomi M
authors.initials: NM
authors.lastname: Hamburg
authors.affiliation: Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University School of Medicine, Boston, MA, USA.
authors.forename: Joseph A
authors.initials: JA
authors.lastname: Vita
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D000328:Adult; D000368:Aged; D001343:Autophagy / Q000187:drug effects*; D015415:Biomarkers / Q000378:metabolism; D016022:Case-Control Studies; D002469:Cell Separation / Q000379:methods; D002478:Cells, Cultured; D003924:Diabetes Mellitus, Type 2 / Q000097:blood / Q000188:drug therapy / Q000473:pathology* / Q000503:physiopathology; D003925:Diabetic Angiopathies / Q000097:blood / Q000473:pathology* / Q000503:physiopathology / Q000517:prevention & control; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D018942:Macrolides / Q000494:pharmacology; D008297:Male; D008875:Middle Aged; D009569:Nitric Oxide / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D013095:Spermidine / Q000494:pharmacology*
chemical_list: D015415:Biomarkers; D018942:Macrolides; D009569:Nitric Oxide; C031080:3-methyladenosine; C040929:bafilomycin A1; C496314:NOS3 protein, human; D052250:Nitric Oxide Synthase Type III; D000241:Adenosine; D013095:Spermidine
keywords: Autophagy; Diabetes mellitus; Endothelial cells