The directed movement of neutrophils is provided by the rapid polymerization of actin with the formation of a protrusion growing forward. In our previous work we observed impaired neutrophil movement for patients with Wiskott-Aldrich syndrome (WAS) compared to healthy donors.In this work, we set out to explain the impairment of neutrophil chemotaxis in patients by observation and computer modeling of the linear growth rates of the anterior pseudopodia. The neutrophil chemotaxis was observed by means of low-angle fluorescent microscopy in parallel-plate flow chambers. The computational model was constructed as a network-like 2D stochastic polymerization of actin guided by the proximity of cell membrane with branching governed by Arp2/3 and WASP proteins.The observed linear velocity of neutrophil pseudopodium formation was 0.22 ± 0.04 μm/s for healthy donors and 0.23 ± 0.08 μm/s for WAS patients. The model described the velocity of the pseudopodium formation for healthy donors well. For the description of WAS patients data, a variation of branching velocity (governed by WASP) by an order of magnitude was applied, which did not significantly alter the linear protrusion growth velocity.We conclude that the proposed mathematical model of neutrophil pseudopodium formation could describe the experimental data well, but the data on overall neutrophil movement could not be explained by the velocities of the pseudopodium growth.
Disruption of adherens junctions (AJs) in endothelial cells by pro‐inflammatory stimuli such as thrombin induces microtubule (MT) destabilization (Gorovoy et al., J Biol Chem. 2005 280: 26533‐42). Here we determined the signaling mechanism regulating MT dynamics by VE‐cadherin homophilic adhesion. We assessed MT dynamics in human microvascular endothelial monolayers by expressing YFP‐tagged End Binding (EB)‐1 protein, which binds preferentially to the growing ends of MTs. Formation of AJs inhibited MT growth by increasing MT catastrophe frequency whereas disruption of AJs by calcium switch or using VE‐cadherin blocking antibody induced MT persistent growth. We used calcium switch model to address the signaling mechanism downstream of VE‐cadherin adhesion in regulating MT dynamics. We observed that disruption of AJs induced the release of calcium from endoplasmic reticulum and activation of protein phosphatase 2B (PP2B). Using PP2B autoinhibitory peptide we demonstrated that PP2B induced de‐phosphorylation of EB3 (but not EB1 or EB2), the accumulation of EB3 at the MT growing ends, and MT growth. We conclude that signaling activated by VE‐cadherin adhesion regulates MT dynamics through the PP2B‐EB3 axis, and may thereby regulate endothelial cells shape and motility.
Rationale The severe acute respiratory syndrome coronavirus 2/coronavirus disease 2019 pandemic has highlighted the serious unmet need for effective therapies that reduce acute respiratory distress syndrome (ARDS) mortality. We explored whether extracellular nicotinamide phosphoribosyltransferase (eNAMPT), a ligand for Toll-like receptor (TLR)4 and a master regulator of innate immunity and inflammation, is a potential ARDS therapeutic target. Methods Wild-type C57BL/6J or endothelial cell (EC)-c NAMPT −/− knockout mice (targeted EC NAMPT deletion) were exposed to either a lipopolysaccharide (LPS)-induced (“one-hit”) or a combined LPS/ventilator (“two-hit”)-induced acute inflammatory lung injury model. A NAMPT-specific monoclonal antibody (mAb) imaging probe ( 99m Tc-ProNamptor) was used to detect NAMPT expression in lung tissues. Either an eNAMPT-neutralising goat polyclonal antibody (pAb) or a humanised monoclonal antibody (ALT-100 mAb) were used in vitro and in vivo . Results Immunohistochemical, biochemical and imaging studies validated time-dependent increases in NAMPT lung tissue expression in both pre-clinical ARDS models. Intravenous delivery of either eNAMPT-neutralising pAb or mAb significantly attenuated inflammatory lung injury (haematoxylin and eosin staining, bronchoalveolar lavage (BAL) protein, BAL polymorphonuclear cells, plasma interleukin-6) in both pre-clinical models. In vitro human lung EC studies demonstrated eNAMPT-neutralising antibodies (pAb, mAb) to strongly abrogate eNAMPT-induced TLR4 pathway activation and EC barrier disruption. In vivo studies in wild-type and EC-c NAMPT −/− mice confirmed a highly significant contribution of EC-derived NAMPT to the severity of inflammatory lung injury in both pre-clinical ARDS models. Conclusions These findings highlight both the role of EC-derived eNAMPT and the potential for biologic targeting of the eNAMPT/TLR4 inflammatory pathway. In combination with predictive eNAMPT biomarker and NAMPT genotyping assays, this offers the opportunity to identify high-risk ARDS subjects for delivery of personalised medicine.
Background: Novel therapeutic targets in radiation pneumonitis (RP), a life-threatening condition, is an important unmet need. Intracellular NAMPT regulates NAD metabolism and is a cancer therapeutic target currently in clinical trials. We have shown that extracellular secreted eNAMPT is a damage-associated molecular pattern protein (DAMP) via ligation of TLR4 and NF-kB inflammatory signalling. Aims: To assess eNAMPT as a potential therapeutic target in preclinical and human RP. Methods: Plasma eNAMPT levels were measured by ELISA. C57/B6 WT and heterozygous NAMPT mice were exposed to a single dose of 20 Gy thoracic radiation and assessed at 4 weeks. A third group consisted of radiated mice received a polyclonal NAMPT-neutralizing antibody (pAb). Standard lung injury assays were performed. Results: Plasma eNAMPT levels in human controls (n=70), subjects with lung cancer (n=42) or breast cancer (n=35), with and without thoracic radiation, revealed significantly increased eNAMPT levels in cancer vs non-cancer patients and in radiated vs non-radiated subjects (p<0.05). Radiated mice demonstrated time-dependent (0-4 wk) increases in lung injury markers reflected by BAL protein and inflammatory cell infiltration that was accompanied by increased tissue NAMPT expression and plasma eNAMPT levels. NAMPT+/- mice were protected against 20Gy injury and weekly intraperitoneal delivery of anti-eNAMPT pAb significantly reduced RP (40-60% decreases in BAL protein, BAL cells, and eNAMPT plasma levels). Conclusion: Extracellular NAMPT is a novel biomarker and attractive therapeutic target in RP and directly contributes to the pathobiology of RP.
Vascular permeability is a hallmark of acute respiratory distress syndrome (ARDS) and ventilator-induced lung injury pathobiology; however, the mechanisms underlying this vascular dysregulation remain unclear, thereby impairing the development of desperately needed effective therapeutics. We have shown that sphingosine-1-phosphate (S1P) and 2-amino-2-(2-[4-octylphenyl]ethyl)-1,3-propanediol (FTY720) analogues are useful tools for exploring vascular barrier regulation mechanisms.
Mechanical ventilation is a life-saving intervention in critically ill patients with respiratory failure due to acute respiratory distress syndrome (ARDS). Paradoxically, mechanical ventilation also creates excessive mechanical stress that directly augments lung injury, a syndrome known as ventilator-induced lung injury (VILI). The pathobiology of VILI and ARDS shares many inflammatory features including increases in lung vascular permeability due to loss of endothelial cell barrier integrity resulting in alveolar flooding. While there have been advances in the understanding of certain elements of VILI and ARDS pathobiology, such as defining the importance of lung inflammatory leukocyte infiltration and highly induced cytokine expression, a deep understanding of the initiating and regulatory pathways involved in these inflammatory responses remains poorly understood. Prevailing evidence indicates that loss of endothelial barrier function plays a primary role in the development of VILI and ARDS. Thus this review will focus on the latest knowledge related to 1) the key role of the endothelium in the pathogenesis of VILI; 2) the transcription factors that relay the effects of excessive mechanical stress in the endothelium; 3) the mechanical stress-induced posttranslational modifications that influence key signaling pathways involved in VILI responses in the endothelium; 4) the genetic and epigenetic regulation of key target genes in the endothelium that are involved in VILI responses; and 5) the need for novel therapeutic strategies for VILI that can preserve endothelial barrier function.
