25-Hydroxycholesterol impairs endothelial function and vasodilation by uncoupling and inhibiting endothelial nitric oxide synthase

Am J Physiol Endocrinol Metab. 2016 Oct 1;311(4):E781-E790. doi: 10.1152/ajpendo.00218.2016. Epub 2016 Sep 6.

Abstract

Endothelial dysfunction is a key early step in atherosclerosis. 25-Hydroxycholesterol (25-OHC) is found in atherosclerotic lesions. However, whether 25-OHC promotes atherosclerosis is unclear. Here, we hypothesized that 25-OHC, a proinflammatory lipid, can impair endothelial function, which may play an important role in atherosclerosis. Bovine aortic endothelial cells were incubated with 25-OHC. Endothelial cell proliferation, migration, and tube formation were measured. Nitric oxide (NO) production and superoxide anion generation were determined. The expression and phosphorylation of endothelial NO synthase (eNOS) and Akt as well as the association of eNOS and heat shock protein (HSP)90 were detected by immunoblot analysis and immunoprecipitation. Endothelial cell apoptosis was monitored by TUNEL staining and caspase-3 activity, and expression of Bcl-2, Bax, cleaved caspase-9, and cleaved caspase-3 were detected by immunoblot analysis. Finally, aortic rings from Sprague-Dawley rats were isolated and treated with 25-OHC, and endothelium-dependent vasodilation was evaluated. 25-OHC significantly inhibited endothelial cell proliferation, migration, and tube formation. 25-OHC markedly decreased NO production and increased superoxide anion generation. 25-OHC reduced the phosphorylation of Akt and eNOS and the association of eNOS and HSP90. 25-OHC also enhanced endothelial cell apoptosis by decreasing Bcl-2 expression and increasing cleaved caspase-9 and cleaved caspase-3 expressions as well as caspase-3 activity. 25-OHC impaired endothelium-dependent vasodilation. These data demonstrated that 25-OHC could impair endothelial function by uncoupling and inhibiting eNOS activity as well as by inducing endothelial cell apoptosis. Our findings indicate that 25-OHC may play an important role in regulating atherosclerosis.

Keywords: 25-hydroxycholesterol; apoptosis; atherosclerosis; endothelial nitric oxide synthase; vasodilation.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Apoptosis / drug effects
  • Cattle
  • Cell Movement / drug effects
  • Cell Proliferation / drug effects
  • Endothelial Cells / drug effects
  • Endothelial Cells / metabolism
  • Endothelium / drug effects*
  • Enzyme Inhibitors / pharmacology*
  • Hydroxycholesterols / pharmacology*
  • In Vitro Techniques
  • Inflammation Mediators / metabolism
  • Nitric Oxide / biosynthesis
  • Nitric Oxide Synthase Type III / antagonists & inhibitors*
  • Rats
  • Rats, Sprague-Dawley
  • Superoxides / metabolism
  • Uncoupling Agents / pharmacology*
  • Vasodilation / drug effects*

Substances

  • Enzyme Inhibitors
  • Hydroxycholesterols
  • Inflammation Mediators
  • Uncoupling Agents
  • Superoxides
  • Nitric Oxide
  • 25-hydroxycholesterol
  • Nitric Oxide Synthase Type III