Neutrophil microvesicles drive atherosclerosis by delivering miR-155 to atheroprone endothelium

Neutrophils are implicated in the pathogenesis of atherosclerosis but are seldom detected in atherosclerotic plaques. We investigated whether neutrophil-derived microvesicles may influence arterial pathophysiology. Here we report that levels of circulating neutrophil microvesicles are enhanced by exposure to a high fat diet, a known risk factor for atherosclerosis. Neutrophil microvesicles accumulate at disease-prone regions of arteries exposed to disturbed flow patterns, and promote vascular inflammation and atherosclerosis in a murine model. Using cultured endothelial cells exposed to disturbed flow, we demonstrate that neutrophil microvesicles promote inflammatory gene expression by delivering miR-155, enhancing NF-κB activation. Similarly, neutrophil microvesicles increase miR-155 and enhance NF-κB at disease-prone sites of disturbed flow in vivo. Enhancement of atherosclerotic plaque formation and increase in macrophage content by neutrophil microvesicles is dependent on miR-155. We conclude that neutrophils contribute to vascular inflammation and atherogenesis through delivery of microvesicles carrying miR-155 to disease-prone regions.British Heart Foundation Programme Grant (CS, PE); British Heart Foundation Project Grants PG/09/067/27901 (AB, VR), PG/13/55/30365 (LW, SF), PG/14/38/30862 (CR, VR), PG/16/44/32146 (JJ, EKT, SF); British Heart Foundation Studentship FS/14/8/30605 (BW, VR); MRC Fellowship MR/K023977/1 (RB); and European Union’s Horizon 2020 Marie Skłodowska-Curie Innovative Training Network, TRAIN 721532 (CN)

Tyrosine Sulfation of Native Mouse Psgl-1 Is Required for Optimal Leukocyte Rolling on P-Selectin In Vivo

We recently demonstrated that tyrosine sulfation is an important contributor to monocyte recruitment and retention in a mouse model of atherosclerosis. P-selectin glycoprotein ligand-1 (Psgl-1) is tyrosine-sulfated in mouse monocyte/macrophages and its interaction with P-selectin is important in monocyte recruitment in atherosclerosis. However, whether tyrosine sulfation is required for the P-selectin binding function of mouse Psgl-1 is unknown. Here we test the function of native Psgl-1 expressed in leukocytes lacking endogenous tyrosylprotein sulfotransferase (TPST) activity.Psgl-1 function was assessed by examining P-selectin dependent leukocyte rolling in post-capillary venules of C57BL6 mice transplanted with hematopoietic progenitors from wild type (WT → B6) or Tpst1;Tpst2 double knockout mice (Tpst DKO → B6) which lack TPST activity. We observed that rolling flux fractions were lower and leukocyte rolling velocities were higher in Tpst DKO → B6 venules compared to WT → B6 venules. Similar results were observed on immobilized P-selectin in vitro. Finally, Tpst DKO leukocytes bound less P-selectin than wild type leukocytes despite equivalent surface expression of Psgl-1.These findings provide direct and convincing evidence that tyrosine sulfation is required for optimal function of mouse Psgl-1 in vivo and suggests that tyrosine sulfation of Psgl-1 contributes to the development of atherosclerosis

Integrin β1 is required for the invasive behaviour but not proliferation of squamous cell carcinoma cells in vivo

Integrin β1 is both overexpressed and in an ‘active' conformation in vulval squamous cell carcinomas (VSCCs) compared to matched normal skin. To investigate the significance of integrin β1 deregulation we stably knocked-down integrin β1 expression in the VSCC cell line A431. In vitro analysis revealed that integrin β1 is required for cell adhesion, cell spreading and invasion. However, integrin β1 is not required for cell growth or activation of FAK and ERK signalling in vitro or in vivo. Strikingly, while control tumours were able to invade the dermis, integrin β1 knockdown tumours were significantly more encapsulated and less invasive

Fibrinogen αC-subregions critically contribute blood clot fibre growth, mechanical stability, and resistance to fibrinolysis

Fibrinogen is essential for blood coagulation. The C-terminus of the fibrinogen α-chain (αC-region) is composed of an αC-domain and αC-connector. Two recombinant fibrinogen variants (α390 and α220) were produced to investigate the role of subregions in modulating clot stability and resistance to lysis. The α390 variant, truncated before the αC-domain, produced clots with a denser structure and thinner fibres. In contrast, the α220 variant, truncated at the start of the αC-connector, produced clots that were porous with short, stunted fibres and visible fibre ends. These clots were mechanically weak and susceptible to lysis. Our data demonstrate differential effects for the αC-subregions in fibrin polymerisation, clot mechanical strength, and fibrinolytic susceptibility. Furthermore, we demonstrate that the αC-subregions are key for promoting longitudinal fibre growth. Together, these findings highlight critical functions of the αC-subregions in relation to clot structure and stability, with future implications for development of novel therapeutics for thrombosis

The expression of E-selectin and chemokines in the cultured human lymphatic endothelium with lipopolysaccharides

This study investigated the expression of selectins and chemokines in cultured human lymphatic endothelial cells stimulated with lipopolysaccharides. In microarray, vascular cell adhesion molecule-1 and intercellular adhesion molecule-1 gene expressions in the lymphatic endothelium with lipopolysaccharides did not change at 0.5 h but increased two- to three-fold at 12 h, whereas E-selectin increased 10-fold at 0.5 h and 68-fold at 12 h compared with untreated cells. The E-selectin mRNA and protein increased in the lymphatic endothelial cells with lipopolysaccharides at more than two-fold levels compared with human umbilical vein endothelial cells. Induction of Cys-Cys chemokine ligand 2, 3, 5, 7, 8 and 20 mRNAs in the lymphatic endothelial cells with lipopolysaccharides was detected in microarray and real-time PCR. The Cys-Cys chemokine ligand 2, 5 and 20 mRNA amounts in cells with high concentration lipopolysaccharides were larger in the lymphatic endothelial cells than in human umbilical vein endothelial cells. The Cys-Cys chemokine ligand 3 and 8 mRNAs were not detected in human umbilical vein endothelial cells. Induction of Cys-X-Cys chemokine ligand 1, 3, 5, 6 and 8 mRNAs was detected in the lymphatic endothelial cells with lipopolysaccharides. The Cys-X-Cys chemokine ligand 3, 5 and 8 mRNA amounts in cells with high concentration lipopolysaccharides were larger in the lymphatic endothelial cells than in human umbilical vein endothelial cells. In conclusion, it was demonstrated that the cultured human lymphatic endothelial cells express E-selectin and phagocyte-attractive chemokine genes