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Effects of fluid shear stress on the anti-inflammatory actions of sulforaphane in human coronary artery endothelial cells

Student thesis: Doctoral ThesisDoctor of Philosophy

Human coronary arteries are prone to develop atherosclerotic lesions, with the pattern of fluid shear stress (FSS) being one of the key factors acting on the endothelial cells (EC) to maintain physiological vascular function. Lesions are more likely to occur at arterial branch points or curvatures as FSS becomes low and oscillatory (OS) in these regions. A deficit in the activation of the transcription factor (TF), nuclear factor (erythroid-derived 2)-like 2 (Nrf2), a ‘master regulator’ of antioxidant genes, results in EC dysfunction, enhanced oxidative stress and expression of pro-inflammatory genes such as the TF activator protein-1 (AP-1) and cell adhesion molecules (CAM) leading to transmigration of monocytes to the intima resulting to the initiation of lesion formation. In contrast, high unidirectional FSS (US) protects vessels against atherogenesis through enhanced activation of Nrf2 in EC which in concert with the TF, Krüppel-like factor 2 and 4 (Klf2/4) act as gatekeepers to maintain redox state and maintain EC in an anti-inflammatory phenotype. The phytonutrient sulforaphane (SFN), has been demonstrated to be atheroprotective via the upregulation of Nrf2 and decreased expression of CAM in EC, however, to date in vitro studies of the actions of SFN in EC have been limited to static cultures.
This study provides the first detailed analysis of the effects of SFN (5 M) treatment in human coronary artery endothelial cells (HCAEC) cultured for up to 24h under well-defined FSS patterns; OS, (±7.5 dynes/cm2, 1Hz) or US (15 dynes/cm2), to investigate changes in key TF and kinase signalling pathways at the level of mRNA and protein expression and phosphorylation. Treatment with SFN for 24h reduced the alignment of EC to the direction of flow following US, and during OS and US, SFN enhanced the expression of the Nrf2 target genes such as NAD(P)H quinone oxidoreductase-1 (NQO1) and heme oxygnenase-1 (HO-1). Despite upregulation of genes involved in the synthesis of the intracellular antioxidant glutathione (GSH) following SFN co-treatment, such as glutamate cysteine-ligase (GCL), intracellular GSH levels were significantly reduced following exposure of EC for 24h to both OS and US. Although US enhanced Nrf2-regulated gene expression, this was paradoxically associated with decreased expression of Klf2/4 target genes such as endothelial nitric oxide synthase (eNOS), argininosuccinate synthase-1 and thrombomodulin. Treatment of EC with SFN during OS suppressed expression of inflammatory genes such as vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP), which was associated with decreased phosphorylation of the AP-1 activator, c-Jun N-terminal kinase (JNK1/2). In summary, this study demonstrates for the first time that treatment of EC exposed to OS can ameliorate deficits in Nrf2 activation and suppress the expression of CAM and chemokines via reduced JNK1/2 phosphorylation. However, SFN treatment of EC exposed to US resulted in a reduction of Klf2/Klf4 signalling. Findings from this study suggest that SFN may be beneficial in reducing pro-inflammatory responses in EC exposed to atherogenic FSS. Although the temporal and spatial effects of SFN on EC exposed to atheroprotective FSS remain to be fully elucidated, it is possible that dietary SFN can ameliorate EC dysfunction through modulating the cross-talk between Nrf2 and Klf2/4 signalling pathways to augment antioxidant gene expression and thereby reduce the incidence of lesion formation.
Original languageEnglish
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Award date2015

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