Polarization of ADAM17-driven EGFR signalling in electric field-guided collective migration of epidermal sheets.

Endogenous electric field is considered to play an important role in promoting collective migration of epidermis to the wound centre. However, most studies are focused on the effect of bioelectric field on the movement and migration of single epithelial cell; the molecular mechanisms about collective migration of epidermal monolayers remain unclear. Here, we found that EFs dramatically promoted the collective migration of HaCaT cells towards the anode, activated the sheddase activity of ADAM17 and increased the phosphorylation level of EGFR. Moreover, EGFR phosphorylation and HB-EGF shedding level were significantly decreased by the ADAM17 inhibitor TAPI-2 or siADAM17 under EFs, which subsequently attenuated the directed migration of HaCaT sheets. Notably, the inhibition of EF-regulated collective migration by siADAM17 was rescued by addition of recombinant HB-EGF. Furthermore, we observed that F-actin was dynamically polarized along the leading edge of the migrated sheets under EFs and that this polarization was regulated by ADAM17/HB-EGF/EGFR signalling. In conclusion, our study indicated that ADAM17 contributed to the collective directional movement of the epidermal monolayer by driving HB-EGF release and activating EGFR under EFs, and this pathway also mediated the polarization of F-actin in migrating sheets, which is essential in directional migration.

Gpbar-1/cAMP/PKA signaling mitigates macrophage-mediated acute cholestatic liver injury via antagonizing NLRP3-ASC inflammasome.

Acute cholestatic liver injury (ACLI) is a disease associated with bile duct obstruction that causes liver inflammation and apoptosis. Although G protein-coupled bile acid receptor1 (Gpbar-1) has diverse metabolic roles, its involvement in ACLI-associated immune activation remains unclear. Liver tissues and blood samples from 20 patients with ACLI and 20 healthy individuals were analyzed using biochemical tests, H&E staining, western blotting, and immunohistochemistry to verify liver damage and expression of Gpbar-1. The expression of Gpbar-1, cAMP/PKA signaling, and the NLRP3 inflammasome was tested in wild-type (WT) and Gpbar-1 knockdown (si-Gpbar-1) mice with ACLI induced by bile duct ligation (BDL) and in primary Kupffer cells (KCs) with or without Gpbar-1-siRNA. The results showed that total bile acids and Gpbar-1 expressions were elevated in patients with ACLI. Gpbar-1 knockdown significantly worsened BDL-induced acute hepatic damage, inflammation, and liver apoptosis in vivo. Knockdown of Gpbar-1 heightened KC sensitivity to lipopolysaccharide (LPS) stimulation. Gpbar-1 activation inhibited LPS-induced pro-inflammatory responses in normal KCs but not in Gpbar-1-knockdown KCs. Notably, NLRP3-ASC inflammasome expression was effectively enhanced by Gpbar-1 deficiency. Additionally, Gpbar-1 directly increased intracellular cAMP levels and PKA phosphorylation, thus disrupting the NLRP3-ASC inflammasome. The pro-inflammatory characteristic of Gpbar-1 deficiency was almost neutralized by the NLRP3 inhibitor CY-09. In vitro, M1 polarization was accelerated in LPS-stimulated Gpbar-1-knockdown KCs. Therapeutically, Gpbar-1 deficiency exacerbated BDL-induced ACLI, which could be rescued by inhibition of the NLRP3-ASC inflammasome. Our study reveal that Gpbar-1 may act as a novel immune-mediated regulator of ACLI by inhibiting the NLRP3-ASC inflammasome.

Optimizing microenvironment by integrating negative pressure and exogenous electric fields via a flexible porous conductive dressing to accelerate wound healing.

Wound healing is a complex and sequential biological process that involves multiple stages. Current treatments for nonhealing or chronic wounds are unsatisfactory as they exert a single effect on one specific activity. Herein, we constructed a silver nanowire (AgNW)-based, three-dimensional (3D), porous foam dressing that is flexible and conductive. This conductive foam dressing was composed of AgNWs modified with a stable hydrophobic coating and porous polyurethane (PU), providing a skeleton to support the 3D conductive networks. The AgNWs-PU foam dressing exhibited favorable biocompatibility, outstanding electrical properties, excellent bending-compression durability, and long-term stability under wet conditions, making it suitable for wound treatment. Via the conductive foam dressing, negative pressure and exogenous wound directional electric fields (EFs) could be integrated for simultaneous implementation, and the artificial jointly constructed microenvironment promoted wound healing in a system. This novel "all-in-one" device presented intrinsic multifunctionality, including the drainage of pus and necrotic tissue, mitigation of inflammation, promotion of cell proliferation, direction of keratinocyte migration, and induction of angiogenesis. An immunohistochemical assay and western blot analysis illustrated that the angiogenesis and cell proliferation pathways in the tissue were significantly activated when this novel therapy was adopted. More importantly, the practical performance of this "all-in-one" device was demonstrated by assessment of full-thickness defect wounds in model pigs. Comparing the percentage of residual wound area after administration of traditional treatment (25.82 ± 3.52%) and the novel treatment (3.07 ± 1.23%) demonstrated the promising applications of this novel treatment in clinical wound healing.

ADAM17 Mediates Hypoxia-Induced Keratinocyte Migration via the p38/MAPK Pathway.

Although hypoxia has been shown to promote keratinocyte migration and reepithelialization, the underlying molecular mechanisms remain largely unknown. ADAM17, a member of the metalloproteinase superfamily, has been implicated in a variety of cellular behaviors such as proliferation, adhesion, and migration. ADAM17 is known to promote cancer cell migration under hypoxia, and whether or how ADAM17 plays a role in hypoxia-induced keratinocyte migration has not been identified. Here, we found that ADAM17 expression and activity were significantly promoted in keratinocytes under hypoxic condition, inhibition of ADAM17 by TAPI-2, or silencing of ADAM17 using small interfering RNA which suppressed the hypoxia-induced migration of keratinocytes significantly, indicating a pivotal role for ADAM17 in keratinocyte migration. Further, we showed that p38/MAPK was activated by hypoxia. SB203580, an inhibitor of p38/MAPK, significantly attenuated the upregulation of ADAM17 as well as the migration of keratinocytes induced by hypoxia. Activation of p38/MAPK by MKK6 (Glu) overexpression, however, had adverse effects. Taken together, our study demonstrated that hypoxia-induced keratinocyte migration requires the p38/MAPK-ADAM17 signal axis, which sheds new light on the regulatory mechanisms of keratinocyte migration. Our study might also help in developing therapeutic strategies to facilitate wound healing in vivo, where cells are migrated in a hypoxic microenvironment.

Changes of Extravascular Lung Water as an Independent Prognostic Factor for Early Developed ARDS in Severely Burned Patients.

An important feature of acute respiratory distress syndrome (ARDS) is fluid lost into the interstitium of lung combined with its compromised reabsorption, resulting in the elevation of extravascular lung water (EVLW). Although ARDS is known as an early, common, and life-threatening complication in major burns, the issue of whether or how the EVLW index (EVLWI) correlates with its prognosis has not been identified yet. In this retrospectively study, 121 severely burned adults with ARDS occurred in 2 weeks postburn were analyzed and divided into two groups: survivors (73 patients) and nonsurvivors (48 patients) according to the 28-day outcome after injury. Compared with nonsurvivors, survivors exhibited bigger EVLWI reduction in day 2 after ARDS onset (ΔEVLWI2), with no differences in ARDS timing and other EVLWI variables. ΔEVLWI2, rather than EVLWI on 2 days after ARDS onset, was identified as an independent prognostic factor even after adjusting other significant factors by Cox proportional hazard analysis. ROC curve analysis showed that ΔEVLWI2 [AUC = 0.723, 95% CI = (0.631-0.816), P < .001] was a relative predictor for survival on 28-day postburn, with a threshold of 1.9 ml/kg (63.0% sensitivity, 77.1% specificity). Kaplan-Meier survival curve analysis confirmed a significantly higher survival rate on 28-day postburn in patients with ΔEVLWI2 > 1.9 ml/kg (log-rank test: χ 2 =14.780, P < .001). Taken together, our study demonstrated that ΔEVLWI2 is an independent prognostic factor for early ARDS in severe burns. ΔEVLWI2 higher than 1.9 ml/kg might predict a higher survival rate in those patients.

CD9 regulates keratinocyte migration by negatively modulating the sheddase activity of ADAM17.

CD9 is a trans-membrane protein, and has recently been implicated in different physiological and cellular processes, such as cell migration and adhesion. According to previous study, down-regulation of CD9 contributes to keratinocyte migration, critical for wound re-epithelialization. Nevertheless, it is widely believed that tetraspanin CD9 does not have ligands or function as the cell surface receptor, rather it is thought to associate with other transmembrane molecules, thereby mediate keratinocyte migration. Little is known about how CD9 associates with transmembrane molecules in migratory keratinocytes. Here, using confocal microscopy, we observed that tetraspanin CD9 and ADAM17 co-localized on the surface of keratinocytes in the course of wound repair in vivo and in vitro. Co-immunoprecipitation experiments demonstrated a direct association between CD9 and ADAM17 in HaCaT cells and C57-MKs. Functional studies revealed that down-regulation or over-expression of CD9 exerted negative regulatory effects on ADAM17 sheddase activity. This activity is involved in CD9-regulated cell motility and migration. Further studies found that ADAM17 inhibitor-TAPI-2 or siADAM17 significantly abolished the enhanced effect of keratinocyte migration induced by CD9 down-regulation. Meanwhile, the sheddase activity of ADAM17 was inhibited by TAPI-2, which decreased this release of AREG and HB-EGF in CD9-silenced HaCat cells and C57-MKs. Importantly, neutralizing antibody against HB-EGF significant weakened keratinocyte migration and motility in CD9-silenced keratinocytes, and the inhibition of CD9-regulated keratinocyte migration by siADAM17 was rescued by addition of recombinant HB-EGF, activating EGFR/ERK pathway. Collectively, our results suggest that ADAM17 sheddase activity is activated by down-regulation of CD9, thereby mediating shedding of HB-EGF and activation of EGFR/ERK signaling, which crucially affects the keratinocyte migration and wound healing.