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Pharmacological inhibition of NOX reduces atherosclerotic lesions, vascular ROS and immune–inflammatory responses in diabetic Apoe −/− mice

Abstract

Aims/hypothesis

Enhanced vascular inflammation, immune cell infiltration and elevated production of reactive oxygen species (ROS) contribute significantly to pro-atherogenic responses in diabetes. We assessed the immunomodulatory role of NADPH oxidase (NOX)-derived ROS in diabetes-accelerated atherosclerosis.

Methods

Diabetes was induced in male Apoe −/− mice with five daily doses of streptozotocin (55 mg kg−1 day−1). Atherosclerotic plaque size, markers of ROS and immune cell accumulation were assessed in addition to flow cytometric analyses of cells isolated from the adjacent mediastinal lymph nodes (meLNs). The role of NOX-derived ROS was investigated using the NOX inhibitor, GKT137831 (60 mg/kg per day; gavage) administered to diabetic and non-diabetic Apoe −/− mice for 10 weeks.

Results

Diabetes increased atherosclerotic plaque development in the aortic sinus and this correlated with increased lesional accumulation of T cells and CD11c+ cells and altered T cell activation in the adjacent meLNs. Diabetic Apoe −/− mice demonstrated an elevation in vascular ROS production and expression of the proinflammatory markers monocyte chemoattractant protein 1, vascular adhesion molecule 1 and IFNγ. Blockade of NOX-derived ROS using GKT137831 prevented the diabetes-mediated increase in atherosclerotic plaque area and associated vascular T cell infiltration and also significantly reduced vascular ROS as well as markers of inflammation and plaque necrotic core area.

Conclusions/interpretation

Diabetes promotes pro-inflammatory immune responses in the aortic sinus and its associated lymphoid tissue. These changes are associated with increased ROS production by NOX. Blockade of NOX-derived ROS using the NOX inhibitor GKT137831 is associated with attenuation of these changes in the immune response and reduces the diabetes-accelerated development of atherosclerotic plaques in Apoe −/− mice.

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Abbreviations

DC:

Dendritic cell

DHE:

Dihydroethidium

MCP-1:

Monocyte chemoattractant protein 1

meLN:

Mediastinal lymph node

NT:

Nitrotyrosine

NOX:

NADPH oxidase

ROS:

Reactive oxygen species

VCAM-1:

Vascular adhesion molecule 1

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Acknowledgements

The authors would like to thank E. Grixti for his expert assistance in measuring metabolic variables, M. Arnstein for assistance with general tissue pathology/immunohistochemistry and K. Gilbert for maintenance of the mice (all affiliated with Baker IDI Heart and Diabetes Institute, Melbourne, VIC, Australia). Appreciation extends to G. Paukovic (Burnet Institute, Melbourne, VIC, Australia) for his expertise in flow cytometry. Kind thanks are also given to A. Bobik (Department of Vascular Biology and Atherosclerosis, Baker IDI Heart and Diabetes Institute) for intellectual contribution to discussions.

Funding

This work was supported by the National Health and Medical Research Council project grant of Australia. KAJD is supported by an NHMRC senior Research Fellowship and MEC is an Australian Fellow for the NHMRC and a JDRF scholar. EDM is supported by a Post-graduate Fellowship (PB 12M 6943) from the National Heart Foundation of Australia.

Duality of interest

CS is a paid employee and owns shares in Genkyotex SA, Geneva, Switzerland. All other authors declare that there is no duality of interest associated with their contribution to this manuscript.

Contribution statement

EDM contributed to the direction of the study, conducted data collection and subsequent analyses and wrote and edited the manuscript. SPG directed the study, conducted data analysis and wrote and edited the manuscript. PC performed and analysed some of the immunohistochemical stains and reviewed the manuscript. CK contributed to collection of meLNs, analysis of some immunohistochemistical stains and reviewed the manuscript. AZ assisted with flow cytometry data collection and analysis and reviewed the manuscript. CS was involved in the conception of the study design, contributed to the protocol for GKT137831 preparation and administration and reviewed and edited the manuscript. RMT and HHHWS assisted with data analysis of oxidative stress variables and also reviewed the manuscript. MEC contributed to data analysis and critical revisions of intellectual content of the manuscript. RS assisted with flow cytometry analysis and edited and reviewed the manuscript. KAJD contributed to the design and supervision of the study, provided intellectual input to discussions and edited and reviewed the manuscript. All authors approved the manuscript for publication.

Author information

Correspondence to K. A. Jandeleit-Dahm.

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Di Marco, E., Gray, S.P., Chew, P. et al. Pharmacological inhibition of NOX reduces atherosclerotic lesions, vascular ROS and immune–inflammatory responses in diabetic Apoe −/− mice. Diabetologia 57, 633–642 (2014). https://doi.org/10.1007/s00125-013-3118-3

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Keywords

  • Atherosclerosis
  • Diabetes
  • Immune system
  • Inflammation
  • NOX