We have made the seminal observation that O-linked ?-N-acetylglucosamine (O-GlcNAc) modification of proteins induced by three distinct and independent stimuli, 17-? estradiol (E2), glucosamine (GlcN) and the selective O-GlcNAcase inhibitor O-(acetamido-2-deoxy-D-glucopyranosylidene) amino-N-phenylcarbamate (PUGNAc), has anti-inflammatory effects in balloon injured rat carotid arteries. These novel observations provide provocative evidence that enhanced O-GlcNAc modification of proteins has anti-inflammatory and vasoprotective effects in the setting of acute endoluminal vascular injury. We have strong evidence that increased protein O-GlcNAc modification in response to GlcN treatment is associated with attenuation of TNF- ?-induced expression of inflammatory mediators in isolated rat aortic smooth muscle cells (RASMCs) in a manner previously reported for E2. We have utilized the TNF-?-treated RASMC as an in vitro model of the acute vascular injury response and have begun to define the mechanisms by which interventions that stimulate protein O-GlcNAc modification, i.e., E2 and GlcN, inhibit inflammatory responses to TNF-?. We have focused on the NF?B signaling pathway, which is known to be activated by both TNF-? and acute vascular injury. Initial experiments demonstrated that GlcN inhibits TNF-?-induced NF?B activation in RASMCs. Subsequent studies showed that pretreatment with GlcN inhibits TNF-?-induced phosphorylation and degradation of I?B? in RASMCs, while E2 pretreatment is associated with an initial reduction, followed by an accelerated reappearance of I?B? in TNF-? treated cells, likely reflecting new protein synthesis mediated by activated NF?B. The current study will test directly the hypothesis that O-GlcNAc modification of proteins, including I?B?, plays a mechanistic role in regulating the inflammatory response to endoluminal arterial injury in vivo and to TNF-? stimulation in isolated RASMCs in vitro.
The Specific Aims are:
Specific Aim 1 : To test the hypothesis that increasing protein O-GlcNAc modification protects arteries from inflammatory stress related to acute endoluminal injury in vivo via inhibition of NF?B signaling.
Specific Aim 2 : To test the hypothesis that increasing protein O-GlcNAc modification inhibits TNF-?-induced inflammatory responses in RASMCs in vitro via inhibition of NF?B signaling and define the precise sites in the NF?B signaling cascade that are responsible for this effect.
Specific Aim 3 : To identify specific protein targets of O-GlcNAc modification in RASMCs that play a functional role in the anti-inflammatory effects of GlcN and E2. Upon successful completion of these Aims, cellular/molecular mechanisms responsible for the anti-inflammatory and vasoprotective actions of O- GlcNAc modification will be elucidated and will be related to the extent of the injury response (i.e., inflammation and neointima formation). We postulate that O-GlcNAc modification represents a novel mechanism of vasoprotection that may lend itself to the development of new strategies for the prevention and treatment of cardiovascular disease.

Public Health Relevance

This proposal will test the hypothesis that a novel mechanism, O-linked acetylglucosamine (O-GlcNAc) modification of proteins, has anti-inflammatory and vasoprotective effects in injured arteries and cytokine- stimulated smooth muscle cells (SMCs) and will identify the specific proteins that are responsible for these protective effects.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Research Project (R01)
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Atherosclerosis and Inflammation of the Cardiovascular System Study Section (AICS)
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OH, Youngsuk
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University of Alabama Birmingham
Internal Medicine/Medicine
Schools of Medicine
United States
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Giordano, Samantha; Zhao, Xiangmin; Chen, Yiu-Fai et al. (2017) Induced Pluripotent Stem Cell-Derived Endothelial Cells Overexpressing Interleukin-8 Receptors A/B and/or C-C Chemokine Receptors 2/5 Inhibit Vascular Injury Response. Stem Cells Transl Med 6:1168-1177
Giordano, Samantha; Zhao, Xiangmin; Xing, Daisy et al. (2016) Targeted delivery of human iPS-ECs overexpressing IL-8 receptors inhibits neointimal and inflammatory responses to vascular injury in the rat. Am J Physiol Heart Circ Physiol 310:H705-15
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Xing, Dongqi; Oparil, Suzanne; Yu, Hao et al. (2012) Estrogen modulates NF?B signaling by enhancing I?B? levels and blocking p65 binding at the promoters of inflammatory genes via estrogen receptor-?. PLoS One 7:e36890
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