Abstract
In addition to cholesterol-lowering properties, statins exhibit lipid-independent immunomodulatory, anti-inflammatory actions. However, high concentrations are typically required to induce these effects in vitro, raising questions concerning therapeutic relevance. We present evidence that endothelial cell sensitivity to statins depends upon shear stress. Using heme oxygenase-1 expression as a model, we demonstrate differential heme oxygenase-1 induction by atorvastatin in atheroresistant compared with atheroprone sites of the murine aorta. In vitro, exposure of human endothelial cells to laminar shear stress significantly reduced the statin concentration required to induce heme oxygenase-1 and protect against H(2)O(2)-mediated injury. Synergy was observed between laminar shear stress and atorvastatin, resulting in optimal expression of heme oxygenase-1 and resistance to oxidative stress, a response inhibited by heme oxygenase-1 small interfering RNA. Moreover, treatment of laminar shear stress-exposed endothelial cells resulted in a significant fall in intracellular cholesterol. Mechanistically, synergy required Akt phosphorylation, activation of Kruppel-like factor 2, NF-E2-related factor-2 (Nrf2), increased nitric-oxide synthase activity, and enhanced HO-1 mRNA stability. In contrast, heme oxygenase-1 induction by atorvastatin in endothelial cells exposed to oscillatory flow was markedly attenuated. We have identified a novel relationship between laminar shear stress and statins, demonstrating that atorvastatin-mediated heme oxygenase-1-dependent antioxidant effects are laminar shear stress-dependent, proving the principle that biomechanical signaling contributes significantly to endothelial responsiveness to pharmacological agents. Our findings suggest statin pleiotropy may be suboptimal at disturbed flow atherosusceptible sites, emphasizing the need for more specific therapeutic agents, such as those targeting Kruppel-like factor 2 or Nrf2.
Highlights
026/20419. □S The on-line version of this article contains supplemental Figs
Heme oxygenase-1 (HO-1) induction was detectable, endothelial cells (EC) located in the lesser curvature of the aorta, which has a high probability of developing lesions, were relatively refractory to atorvastatin treatment (Fig. 1B)
We have explored the hypothesis that laminar shear stress (LSS) conditioning of endothelium enhances the cytoprotective effects of statins, using HO-1 induction as a model
Summary
Reagents—Actinomycin D, hydrogen peroxide (H2O2), paraformaldeyde, Triton X-100, trypan blue, and anti-␣-tubulin antibody were from Sigma. Endothelial Cell Exposure to Shear Stress—Human umbilical vein EC (HUVEC) and human aortic EC (purchased from Promocell, Heidelberg, Germany) were cultured as described [20]. After 12 h, statin or vehicle control was added to the culture medium of static cells or to the medium in the flow apparatus via the injection port, whereas EC remained under conditions of continuous LSS. Changes in the expression of HO-1 in murine aortic EC located in regions of the lesser curvature exposed to disturbed flow and both the greater curvature and descending aorta exposed to laminar flow were quantified as described [27]. HO-1 protein expression was quantified by image analysis of fluorescence intensity in 100 cells in at least 3 distinct sites using Image J software. Differences were considered significant at p values of Ͻ0.05
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