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Leukocyte transendothelial migration (TEM) is absolutely fundamental to the inflammatory response, and involves initial pseudopod protrusion and subsequent polarised migration across inflamed endothelium. Ezrin/radixin/moesin (ERM) proteins are expressed in leukocytes and mediate cell shape changes and polarity. The spatio-temporal organisation of ERM proteins with their targets, and their individual contribution to protrusion during TEM, has never been explored. Here, we show that blocking binding of moesin to phosphatidylinositol 4,5-bisphosphate (PIP2) reduces its C-terminal phosphorylation during monocyte TEM, and that on–off cycling of ERM activity is essential for pseudopod protrusion into the subendothelial space. Reactivation of ERM proteins within transmigrated pseudopods re-establishes their binding to targets, such as L-selectin. Knockdown of ezrin, but not moesin, severely impaired the recruitment of monocytes to activated endothelial monolayers under flow, suggesting that this protein plays a unique role in the early recruitment process. Ezrin binds preferentially to L-selectin in resting cells and during early TEM. The moesin–L-selectin interaction increases within transmigrated pseudopods as TEM proceeds, facilitating localised L-selectin ectodomain shedding. In contrast, a non-cleavable L-selectin mutant binds selectively to ezrin, driving multi-pseudopodial extensions. Taken together, these results show that ezrin and moesin play mutually exclusive roles in modulating L-selectin signalling and shedding to control protrusion dynamics and polarity during monocyte TEM., Summary: Ezrin and moesin co-ordinate binding to L-selectin in monocytes to, respectively, regulate pseudopod protrusion and ectodomain shedding during transendothelial migration.

Endothelial cells (EC) in athero-prone regions and athero-protected regions respectively experience low, oscillatory (OS) and high unidirectional (US) fluid shear stress (FSS). Reduced activity of the antioxidant transcription factor Nrf2 in EC exposed to OS may contribute to oxidative stress and lesion formation. Our previous work using human umbilical vein EC (HUVEC) demonstrated that cells exposed to US (15 dyn/cm2) exhibited enhanced Nrf2 expression and activation of downstream antioxidant genes compared to cells exposed to OS (±5 dyn/cm2), concomitant with reduced expression of its competitive inhibitor Bach1. Furthermore, miRNA-320a levels are elevated in HUVEC exposed to US whilst miRNA-21-3p is enhanced by OS. We show that EaHy926 cells cultured under FSS exhibit comparable Nrf2 responses and miRNA patterns to HUVEC. EC were transfected with miRNA-21-3p and miRNA-320a mimics, and effects on protein and mRNA expression of Nrf2, its regulatory proteins and downstream targets were assessed. Bach1 protein levels were enhanced by miR-21-3p and reduced by miR-320a. Our findings suggest that these mechanosensitive miRNAs may regulate EC redox phenotype by modulating the Nrf2 antioxidant signaling pathway in response to FSS.

Atherosclerotic lesions develop in regions of the vasculature with complex fluid shear stress (FSS) patterns. Endothelial cells (EC) in atheroprone and atheroprotected regions are exposed to low oscillatory (OS) or high unidirectional (US) shear stresses respectively. Oxidative stress and altered activity of the transcription factor Nrf2 have been shown to contribute to atherogenesis. Using in vitro culture of human umbilical vein EC (HUVEC) under FSS, we have confirmed that expression and nuclear translocation of Nrf2 are enhanced by US (15 dyn/cm2) compared with OS (±5 dyn/cm2), enhancing expression of antioxidant genes such as heme oxygenase-1 (HO-1) and glutathione levels. This study investigates whether mechanosensitive microRNAs may contribute to FSS-dependent Nrf2 regulation. A screen of microRNAs known or predicted to target antioxidant genes revealed that microRNAs miR-21-3p, miR-320a, and miR-409-5p are significantly differentially regulated in HUVEC exposed to OS or US for 24 hours. Overexpression of miR-320a in static HUVEC using miRNA mimics significantly enhanced protein expression of HO-1, recapitulating observations in EC under US. This study suggests that modulation of microRNAs may ameliorate deficits in redox signalling in EC under atheroprone FSS.