Abstract
Activation of the nuclear factor erythroid 2–related factor 2 (Nrf2) pathway is critical for vascular endothelial redox homeostasis in regions of high, unidirectional shear stress (USS), however the underlying mechanosensitive mediators are not fully understood. The endothelial glycocalyx is disrupted in arterial areas exposed to disturbed blood flow that also exhibit enhanced oxidative stress leading to atherogenesis. We investigated the contribution of glycocalyx sialic acids (SIA) to Nrf2 signaling in human endothelial cells (EC) exposed to atheroprotective USS or atherogenic low oscillatory shear stress (OSS). Cells exposed to USS exhibited a thicker glycocalyx and enhanced turnover of SIA which was reduced in cells cultured under OSS. Physiological USS, but not disturbed OSS, enhanced Nrf2-mediated expression of antioxidant enzymes, which was attenuated following SIA cleavage with exogenous neuraminidase. SIA removal disrupted kinase signaling involved in the nuclear accumulation of Nrf2 elicited by USS and promoted mitochondrial reactive oxygen species accumulation. Notably, knockdown of the endogenous sialidase NEU1 potentiated Nrf2 target gene expression, directly implicating SIA in regulation of Nrf2 signaling by USS. In the absence of SIA, deficits in Nrf2 responses to physiological flow were also associated with a pro-inflammatory EC phenotype. This study demonstrates that the glycocalyx modulates endothelial redox state in response to shear stress and provides the first evidence of an atheroprotective synergism between SIA and Nrf2 antioxidant signaling. The endothelial glycocalyx therefore represents a potential therapeutic target against EC dysfunction in cardiovascular disease and redox dyshomeostasis in ageing.
Highlights
Vascular endothelial cell defences against oxidative stress are coordinated by the transcription factor nuclear factor erythroid 2–related factor 2 (Nrf2), which modulates antioxidant gene expression through binding to DNA sequences termed antioxidant response elements (ARE) [1]
Human umbilical vein endothelial cells (HUVEC) maintained in culture have diminished GCX thickness compared to the umbilical vein in vivo [36], multiple studies have demonstrated the vasoprotective properties of the GCX in vitro
We provide novel evidence that in the absence of sialic acids (SIA), endothelial cells (EC) cultured under unidirectional shear stress (USS) have impaired endothelial nitric oxide synthase (eNOS) phosphorylation, enhanced levels of mitochondrial reactive oxygen species (ROS) and the proinflammatory marker vascular cell adhesion molecule 1 (VCAM-1), phenotypically resembling EC exposed to pro-atherogenic oscillatory shear stress (OSS)
Summary
Vascular endothelial cell defences against oxidative stress are coordinated by the transcription factor Nrf, which modulates antioxidant gene expression through binding to DNA sequences termed antioxidant response elements (ARE) [1]. Proteasomal degradation of constitutively synthesised Nrf is mediated by its cytosolic redox-sensitive partner Kelch-like ECH-associated protein 1 (Keap-1) [2]. Cytotoxic insults such as electrophiles and xenobiotics disrupt this interaction [3, 4], allowing Nrf to accumulate in the nucleus where it promotes the transcription of genes encoding antioxidant, phase II detoxifying and glutathione synthesising enzymes to restore redox balance [5]. Activation of the Nrf pathway by USS can be mediated by kinase signaling events [14, 15] and is primed by shear-sensitive expression of Krüppellike factor 2 (Klf2) [16], responsible for transcriptional programing of endothelial atheroprotection [17]. Nrf stabilization and nuclear translocation in response to OSS does not promote ARE-dependent gene transcription [12] due to additional epigenetic regulation by histone deacetylases and mechanosensitive microRNAs [6]
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