The kinetics of E-selectin- and P-selectin-induced intermediate activation of integrin αLβ2 on neutrophils

ABSTRACT Selectins and integrins are key players in the adhesion and signaling cascade that recruits leukocytes to inflamed tissues. Selectin binding induces β2 integrin binding to slow leukocyte rolling. Here, a micropipette was used to characterize neutrophil adhesion to E-selectin and intercellular adhesion molecule-1 (ICAM-1) at room temperature. The time-dependent adhesion frequency displayed two-stage kinetics, with an E-selectin-mediated fast increase to a low plateau followed by a slow increase to a high plateau mediated by intermediate-affinity binding of integrin αLβ2 to ICAM-1. The αLβ2 activation required more than 5 s contact to E-selectin and spleen tyrosine kinase (Syk) activity. A multi-zone channel was used to analyze αLβ2 activation by P-selectin in separate zones of receptors or antibodies, finding an inverse relationship between the rolling velocity on ICAM-1 and P-selectin dose, and a P-selectin dose-dependent change from bent to extended conformations with a closed headpiece that was faster at 37°C than at room temperature. Activation of αLβ2 exhibited different levels of cooperativity and persistent times depending on the strength and duration of selectin stimulation. These results define the precise timing and kinetics of intermediate activation of αLβ2 by E- and P-selectins.

An Investigation on Starvation Onset of Rough Surface Line Contacts

Utilizing a computational approach, this study quantifies the onset of lubrication starvation for line contacts of rough surfaces operating under typical ranges of automotive gearing applications. The response parameter is selected as the critical film thickness supply, at which starvation initiates. The potential influential parameters (predictors) considered include normal force density, rolling velocity, sliding, lubricant viscosity, and surface roughness amplitude. A non-Newtonian thermal mixed lubrication model is employed to determine the critical lubricant supply under various operating and surface roughness conditions. General linear regression is implemented to reach an easy-to-use equation (R-squared value higher than 97%), facilitating the quantification of starvation dependence on the predictors that are statistically significant.

Mucin-Like Domain of Mucosal Addressin Cell Adhesion Molecule-1 Facilitates Integrin α4β7-Mediated Cell Adhesion Through Electrostatic Repulsion

The homing of lymphocytes from blood to gut-associated lymphoid tissue is regulated by interaction between integrin α4β7 with mucosal vascular addressin cell adhesion molecule 1 (MAdCAM-1) expressed on the endothelium of high endothelial venules (HEVs). However, the molecular basis of mucin-like domain, a specific structure of MAdCAM-1 regulating integrin α4β7-mediated cell adhesion remains obscure. In this study, we used heparan sulfate (HS), which is a highly acidic linear polysaccharide with a highly variable structure, to mimic the negative charges of the extracellular microenvironment and detected the adhesive behaviors of integrin α4β7 expressing 293T cells to immobilized MAdCAM-1 in vitro. The results showed that HS on the surface significantly promoted integrin α4β7-mediated cell adhesion, decreased the percentage of cells firmly bound and increased the rolling velocities at high wall shear stresses, which was dependent on the mucin-like domain of MAdCAM-1. Moreover, breaking the negative charges of the extracellular microenvironment of CHO-K1 cells expressing MAdCAM-1 with sialidase inhibited cell adhesion and rolling velocity of 293T cells. Mechanistically, electrostatic repulsion between mucin-like domain and negative charges of the extracellular microenvironment led to a more upright conformation of MAdCAM-1, which facilitates integrin α4β7-mediated cell adhesion. Our findings elucidated the important role of the mucin-like domain in regulating integrin α4β7-mediated cell adhesion, which could be applied to modulate lymphocyte homing to lymphoid tissues or inflammatory sites.

Aquaporin-1: a major modulator of aging-mediated platelet/endothelial dysfunction in atherothrombosis

Background Aging is associated with development of cardiovascular diseases, including atherothrombosis. Aquaporin-1 (AQP1) is a water channel that also transports hydrogen peroxide (H2O2). Regarding to the role of oxidative stress in atherothrombosis, we hypothesized that AQP1 modulates aging-associated platelet/endothelial dysfunction. Methods Human aortic endothelial cells (HAEC) from passages 5 (young) to 15 (senescent/old) were subjected to fluorescent immunocytochemistry to detect AQP1 protein expression and subcellular localization. The cells were also probed for AQP1 and the phospho/total proteins (AMPK, acetyl-coA-carboxylase (ACC), caveolin-1 and eNOS) for studying the signal transduction by immunoblotting. The endothelial cells were transfected with constructs containing H2O2 biosensor HyPer targeted to cell nucleus or cytosol followed by fluorescence imaging. The transcriptional levels of pro-inflammatory/pro-atherogenic vs. anti-inflammatory/atheroprotective genes in the cells were assessed by qRT-PCR. Human blood samples were taken and treated with or without AQP1 inhibitor (Bacopaside II, 10 μM) to examine the platelet adhesion and rolling velocity on vWF under high shear flow (100 dyn/cm2). Also, platelet aggregation in response to collagen (2 μM), ADP (1 μM) and TRAP (1 μM) were recorded. Results First, the senescence of HAEC was adjusted by a significant increase in β-galactosidase activity from passage 5 to 15. AQP1 immunofluorescence showed a remarkable increase in the young (P.5) compared to senescent (P.15) cells. Immunoblot analyses showed that aging leads to significant increases in AQP1 intensity and phosphorylation of caveolin-1 (Tyr14) and ACC (Ser79), along with decreases in phosphorylation of eNOS (Ser1177) and AMPK (Thr172) (p<0.01, n>6). Fluorescence imaging documented a robust H2O2 production in the senescent endothelial cell cytosol, but not nucleus, and activated TNF-α gene, whereas the transcription of hemoxygenase-1 gene enhanced in the young cells (p<0.01). AQP1 inhibition reduced platelet adhesion and thrombus formation, and elevated platelet rolling velocity on vWF under shear flow (p<0.01). Also, a decrease was found in platelet aggregation in response to AQP1 inhibition (p<0.05). Conclusion These studies, for the first time, demonstrate that aging induces AQP1 expression in endothelial cells and platelets, and modulates the dephosphorylation of AMPK/eNOS. These may lead to platelet/endothelial dysfunction and production of pro-coagulant/pro-inflammatory factors via ACC activation. Therefore, AQP1 inhibition could potentially be exploited as a therapeutic strategy for improving age-related atherothrombosis. Figure 1. Aging increases AQP1 expression in HAEC that leads to dephosphorylation of AMPK and eNOS. AQP inhibition also improves platelet function. *p<0.05, **p<0.01 compared to control. Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): Swiss National Science Foundation (SNSF)

A Transient Mixed Elastohydrodynamic Lubrication Model for Helical Gear Contacts

In this study, a non-Newtonian, transient, isothermal, mixed elastohydrodynamic lubrication (EHL) model is proposed for helical gear contacts. The model accounts for nonelliptical contacts subject to spatially varying sliding and rolling velocity fields that are not aligned with any principal axis of the contact region, which is the case for helical gear contacts. The time-varying changes pertaining to key contact parameters and relative motion of roughness profiles on mating tooth surfaces are captured simultaneously to follow the contact from the root to the tip of a tooth while accounting for the transient effect due to relative motions of the roughness profiles. Actual tooth load distributions, contact kinematics, and compliances of helical gear contacts are provided to this model by an existing helical gear load distribution model. Measured three-dimensional roughness profiles covering the entire meshing zone are incorporated in the analyses to investigate its impact on the EHL conditions as well as mechanical power loss. Results of a parametric sensitivity study are presented to demonstrate the influence of operating conditions and surface roughness on the EHL behavior and the resultant gear mesh mechanical power loss of an example helical gear pair. The accuracy of the proposed mixed-EHL model is assessed by comparing the mechanical power loss predictions to available experimental results.

PREDICTION OF OIL FILM THICKNESS IN TROCHOIDAL PUMP

A model for predicting the oil film thickness between the contact surfaces of the gerotor pump teeth is given in this paper. Gerotor pumps are rotary pumps with inner trochoidal gearing. The working process of the pump is followed by mechanical losses due to friction that occurs between the coupled surfaces. In order to reduce these losses and avoid wearing of the teeth’s active surfaces, it is necessary to provide a minimum thickness of the oil film between the contact surfaces. The minimum thickness of the oil film depends on a large number of different factors, but in this paper for its determination, the Hamrock and Dowson empirical equation is used. Previously, the mathematical model that follows the change of relative velocities in contact, sliding velocity, rolling velocity and contact force at the contact points of the coupled teeth profiles is developed. The influence of different parameters on the lubrication regime has been analyzed, and it has been shown that the trohydraulic pump operates most with the limit EHD lubrication regime. The most unfavorable period occurs when the resulting rolling velocity decreases significantly, and this period dominates during coupling. The results of this research provide guidelines for the design of the tooth profile’s shape and the choice of operating parameters of the pump with high reliability.

Empagliflozin Ameliorates Leukocyte – Endothelium Cell Interactions and Inflammation in Type 2 Diabetic Patients

Background: SGLT2 inhibitors (iSGLT2) such as empagliflozin can reduce cardiovascular risk in patients with type 2 diabetes, but the underlying molecular mechanisms are yet to be determined. In the present study we evaluate the effects of empagliflozin on anthropometric and endocrine parameters, leukocyte-endothelium interactions, adhesion molecules and NFkB-p65 transcription factor expression. Methods: Eighteen patients with type 2 diabetes were recruited for the study. Patients received 10 mg/day of empagliflozin according to standard clinical protocols and were followed-up during a 24-week period. Anthropometric and analytical measurements were performed at baseline, 12-weeks and 24-weeks. Interactions between polymorphonuclear leukocytes and human umbilical vein endothelial cells (HUVECs), serum levels of adhesion molecules (P-selectin, VCAM-1 and ICAM-1) and NFkB-p65 protein levels were measured. Results: We observed a decrease in body weight, BMI and HbA1C levels from 12 weeks of treatment, which had become more pronounced at 24 weeks and was accompanied by a significant reduction in waist circumference, glucose, and hs-CRP levels. Leukocyte-endothelium interactions were reduced due to an enhancement of leukocyte rolling velocity from 12 weeks onwards, together with a significant decrease in leukocyte rolling flux and adhesion at 24 weeks. Accordingly, a significant decrease in ICAM-1 levels and NFkB-p65 expression were observed. Conclusions: Empagliflozin reduced leukocyte-endothelium interactions, adhesion molecules and NFkB-p65 expression in type 2 diabetic patients after 24 weeks of treatment.

Droplet Rolling and Spinning in V-Shaped Hydrophobic Surfaces for Environmental Dust Mitigation

The motion of a water droplet in a hydrophobic wedge fixture was examined to assess droplet rolling and spinning for improved dust mitigation from surfaces. A wedge fixture composed of two inclined hydrophobic plates had different wetting states on surfaces. Droplet rolling and spinning velocities were analyzed and findings were compared with the experiments. A wedge fixture was designed and realized using a 3D printing facility and a high speed recording system was adopted to evaluate droplet motion in the wedge fixture. Polycarbonate sheets were used as plates in the fixture, and solution crystallization and functionalized silica particles coating were adopted separately on plate surfaces, which provided different wetting states on each plate surface while generating different droplet pinning forces on each hydrophobic plate surface. This arrangement also gave rise to the spinning of rolling droplets in the wedge fixture. Experiments were extended to include dust mitigation from inclined hydrophobic surfaces while incorporating spinning- and rolling droplet and rolling droplet-only cases. The findings revealed the wedge fixture arrangement resulted in spinning and rolling droplets and spinning velocity became almost 25% of the droplet rolling velocity, which agrees well with both predictions and experiments. Rolling and spinning droplet gave rise to parallel edges droplet paths on dusty hydrophobic surfaces while striations in droplet paths were observed for rolling droplet-only cases. Spinning and rolling droplets mitigated a relatively larger area of dust on inclined hydrophobic surfaces as compared to their counterparts corresponding to rolling droplet-only cases.

A Strain-Based Intelligent Tire to Detect Contact Patch Features for Complex Maneuvers

Tires are essential components of vehicles and are able to transmit traction and braking forces to the contact patch, contribute to directional stability, and also help to absorb shocks. If these components can provide information related to the tire–road interaction, vehicle safety can be increased. This research is focused on developing the tire as an active sensor capable to provide its functional parameters. Therefore, in this work, we studied strain-based measurements on the contact patch to develop an algorithm to compute the wheel velocity at the contact point, the effective rolling radius and the contact length on dynamic situations. These parameters directly influence the dynamics of wheel behavior which nowadays is not clearly defined. Herein, hypotheses have been assumed based on previous studies to develop the algorithm. The results expose to view an experimental test regarding influence of the tire operational condition (slip angle, vertical load, and rolling velocity) onto the computed parameters. This information is used to feed a fuzzy logic system capable of estimating the effective radius and contact length. Furthermore, a verification process has been carried out using CarSim simulation software to get the inputs for the fuzzy logic system at complex maneuvers.