ATP release and P2Y receptor signalling are essential for keratinocyte galvanotaxis

Repair to damaged tissue requires directional cell migration to heal the wound. Immediately upon wounding an electrical guidance cue is created with the cathode of the electric field (EF) located at the center of the wound. Previous research has demonstrated directional migration of keratinocytes towards the cathode when an EF of physiological strength (100–150 mV/mm) is applied in vitro, but the “sensor” by which keratinocytes sense the EF remains elusive. Here we use a customised chamber design to facilitate the application ofa direct current (DC) EF of physiological strength (100 mV/mm) to keratinocytes whilst pharmacologically modulating the activation ofboth connexin hemichannels and purinergic receptorsto determine their role inEF-mediated directional keratinocyte migration, galvanotaxis. In addition, keratinocytes were exposed to DiSCAC2(3) dye to visualize membrane potential changes within the cell upon exposure to the applied DC EF. Here we unveil ATP-medicated mechanisms that underpin the initiation of keratinocyte galvanotaxis. The application of a DC EF of 100 mV/mm releases ATP via hemichannels activatinga subset of purinergic P2Y receptors, locally, to initiate the directional migration of keratinocytes towards the cathodein vitro, the center of the woundin vivo. The delineation of the mechanisms underpinning galvanotaxis extends our understanding of this endogenous cue and will facilitate the optimization and wider use of EF devices for chronic wound treatment. This article is protected by copyright. All rights reserve

Beta-Adrenoceptor Activation Reduces Both Dermal Microvascular Endothelial Cell Migration Via a cAMP-Dependent Mechanism and Wound Angiogenesis.

Angiogenesis is an essential process during tissue regeneration; however, the amount of angiogenesis directly correlates with the level of wound scarring. Angiogenesis is lower in scar-free fetal wounds while angiogenesis is raised and abnormal in pathophysiological scarring such as hypertrophic scars and keloids. Delineating the mechanisms that modulate angiogenesis and could reduce scarring would be clinically useful. Beta-adrenoceptors (β-AR) are G protein-coupled receptors expressed on all skin cell-types. They play a role in wound repair but their specific role in angiogenesis is unknown. In this study, a range of in vitro assays (single cell migration, scratch wound healing, ELISAs for angiogenic growth factors and tubule formation) were performed with human dermal microvascular endothelial cells (HDMEC) to investigate and dissect mechanisms underpinning β-AR-mediated modulation of angiogenesis in chick chorioallantoic membranes (CAM) and murine excisional skin wounds. β-AR activation reduced HDMEC migration via cAMP-dependent and PKA-independent mechanisms as demonstrated through use of an EPAC agonist that auto-inhibited the cAMP-mediated β-AR transduced reduction in HDMEC motility; a PKA inhibitor was, conversely, ineffective. ELISA studies demonstrated that β-AR activation reduced pro-angiogenic growth factor secretion from HDMECs (fibroblast growth factor 2) and keratinocytes (vascular endothelial growth factor A) revealing possible β-AR-mediated autocrine and paracrine anti-angiogenic mechanisms. In more complex environments, β-AR activation delayed HDMEC tubule formation and decreased angiogenesis both in the CAM assay and in murine excisional skin wounds in vivo. β-AR activation reduced HDMEC function in vitro and angiogenesis in vivo; therefore, β-AR agonists could be promising anti-angiogenic modulators in skin

CXCL8 and GM-CSF induce the phosphorylation of Akt.

<p>Neutrophils were treated with wortmannin (50nM) or the PI3K (1M), (10M) and (10μM) selective inhibitors for 2 min in combination with 500ng/ml of CXCL8 (a,b) or 50ng/ml GM-CSF (c,d). Western blots were carried out using the antibodies Phosphorylated Akt and total Akt (a,c). Densitometry was performed on the western blot films (b,d). The western blots shown are an example of 1 of the 3 carried out which all demonstrated the same pattern. In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116250#pone.0116250.g007" target="_blank">Fig. 7b</a> CXCL8+DMSO significantly increased phosphorylation of Akt compared to the unstimulated control (p<0.05) and PIK-75 (PI3kinase α) caused significant inhibition of phosphorylation (p<0.05). In <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116250#pone.0116250.g007" target="_blank">Fig. 7d</a> the PI3kinase δ inhibitor PIK-294 significantly inhibited GM-CSF+DMSO mediated phosphorylation (p<0.05)</p

Neutrophils express the class I PI3K catalytic subunits α,δ and γ.

<p>RNA was isolated from unstimulated neutrophils. RT-PCR was carried out using primers for each of the four class I PI3-kinase catalytic isoforms and β-actin as a control. n = 1 representative of three experiments. The identity of each of the bands was confirmed by excising the band and sequencing the DNA product.</p

Summary of the degree of inhibition obtained with the PI3 kinase inhibitors on CXCL-8 and GMCSF induced neutrophil migration.

<p>% inhibition</p><p>+++ ~100%</p><p>++ >50%</p><p>- Not significant</p><p>ND: Not done</p><p>Summary of the degree of inhibition obtained with the PI3 kinase inhibitors on CXCL-8 and GMCSF induced neutrophil migration.</p

Neutrophil migration is dose dependent in both the gradient and non-gradient assays.

<p>a) migration in response to CXCL8, *significantly greater as compared to control cells, p<0.05, n = 3 except for non gradient control and 100ng/ml where n = 5. b) The direction of movement for each neutrophil in the non gradient and gradient assay in response to 100ng/ml of CXCL8 is illustrated in the vector diagrams which show chemokinetic migration in the non-gradient assay and a chemotactic pattern of migration in the gradient assay. c) Neutrophil migration to GM-CSF is dose dependent in both the non gradient and gradients assays. *significantly greater as compared to control cells, p<0.05, n = 3 except for gradient control and 50ng/ml where n = 7 and 6 respectively. d). The direction of movement for each neutrophil in the non gradient and gradient assay in response to 50ng/ml of GM-CSF is illustrated in the vector diagrams, These show that the pattern of migration to GM-CSF was chemokinetic in both the gradient and non-gradient assays.</p

An antagonist of PI3Kγ inhibits CXCL8 mediated chemokinetic but not chemotactic migration and GM-CSF mediated chemokinetic migration at a sub-optimal concentration of GM-CSF.

<p>Neutrophils were treated with 10μM PI3Kγ selective inhibitor for 30 mins prior to the addition of CXCL8 (100ng/ml, a) or GM-CSF (0.5ng/ml, 50ng/ml, b). Data shown are mean ±SEM (n = 3) except for non gradient CXCL8 where n = 4 and GM-CSF where n = 6 ***p<0.001.</p

An antagonist of PI3Kα inhibits CXCL8 induced chemotactic migration in a dose and time dependent manner.

<p>a) Neutrophils were pre-treated for 30 mins with varying concentrations (from 0.1μM to 10μM) of the PI3Kα selective inhibitor PIK75, and then stimulated with 100ng/ml CXCL8 in the gradient assay. b) Neutrophils were pre-treated with 1μM of PIK-75 for 30 minutes or 2 hours, prior to construction of the migration gel and the addition of 100ng/ml of CXCL8 in the gradient assay. Results are shown as mean ±SEM; n = 4, except for CXCL8, 1μM and the corresponding DMSO control where n = 11 ***p<0.001, **p<0.01. <b>Antagonists of PI3Kδ and α have additive effects on inhibition of CXCL8 mediated neutrophil chemotaxis.</b> Neutrophils were pre-treated for 30 mins with either 2μM of the PI3Kα selective inhibitor, PIK-75, 2μM of the PI3Kδ selective inhibitor, PIK-274 or 1μM of each and then stimulated with 100ng/ml CXCL8. Results are shown as mean ±SEM (n = 4) *p<0.05. (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116250#pone.0116250.g006" target="_blank">Fig. 6c</a>)</p

Wortmannin inhibits both CXCL8 and GM-CSF-mediated neutrophil migration.

<p>Neutrophils were pre-treated for 30 mins with 50nM wortmannin, and then stimulated with a) CXCL8 (100ng/ml) or b) GM-CSF (0.5ng/ml, 50ng/ml). Results are shown as mean ±SEM (n = 7) except for DMSO controls and 0.5ng/ml GM-CSF where n = 3, ***p<0.001.</p

An antagonist of PI3Kδ inhibits both chemotactic and chemokinetic migration mediated by CXCL8 and GM-CSF-.

<p>Neutrophils were treated with 1μM and 10μM of the PI3Kδ selective inhibitor PIK-274 for 30 mins prior to the addition of CXCL8 (100ng/ml, a) or 0.5ng/ml GM-CSF (b). Data shown are mean ±SEM (n = 3) except for gradient DMSO and 1μM of PIK-274 where n = 8. *p<0.05, ***p<0.001.</p