Stress-Mediated Increases in Systemic and Local Epinephrine Impair Skin Wound Healing: Potential New Indication for Beta Blockers

Rivkah Isseroff and colleagues describe how stress-induced elevation of epinephrine levels can impair the healing of burns in mice and suggest that β2 adrenergic receptor antagonists may have a role in improving skin wound repair

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

A pulsed current electric field alters protein expression creating a wound healing phenotype in human skin cells

Aim: Pulsed current (PC) electric field (EF) devices promote healing in chronic wounds but the underpinning mechanisms are largely unknown. The gap between clinical evidence and mechanistic understanding limits device uptake in clinics. Materials & methods: Migration, proliferation and gene/protein expression profiles were investigated in the presence/absence of PCEF, in skin: keratinocytes (NHK); dermal fibroblasts (HDF); dermal microvascular endothelial cells (HDMEC) and macrophages (THP-1). Results: While PCEF had little effect on migration or proliferation, it significantly altered the expression of 31 genes and the secretion of 7 pro-angiogenic and pro-regenerative growth factors using ELISAs. Conclusion: PCEF significantly altered skin cell genomes/proteomes which provides some evidence of how PCEF devices promote healing of chronic wounds

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

␤2-Adrenergic Receptor Signaling Mediates Corneal Epithelial Wound Repair

PURPOSE. ␤-Adrenergic receptor (AR) antagonists are frequently prescribed ophthalmic drugs, yet previous investigations into how catecholamines affect corneal wound healing have yielded conflicting results. With the use of an integrated pharmacologic and genetic approach, the authors investigated how the ␤-AR impacts corneal epithelial healing. METHODS. Migratory rates of cultured adult murine corneal epithelial (AMCE) cells and in vivo corneal wound healing were examined in ␤2-AR ϩ/ϩ and ␤2-AR Ϫ/Ϫ mice. Signaling pathways were evaluated by immunoblotting. RESULTS. The ␤-AR agonist isoproterenol decreased AMCE cell migratory speed to 70% of untreated controls, and this was correlated with a 0.60-fold decrease in levels of activated phospho-ERK (P-ERK). Treatment with the ␤-AR antagonist (timolol) increased speed 33% and increased P-ERK 2.4-fold (P Ͻ 0.05). The same treatment protocols had no effect on AMCE cells derived from ␤2-AR Ϫ/Ϫ mice; all treatment groups showed statistically equivalent migratory speeds and ERK phosphorylation. In ␤2-AR ϩ/ϩ animals, the ␤-AR agonist (isoproterenol) delayed the rate of in vivo corneal wound healing by 79%, whereas ␤-AR antagonist (timolol) treatment increased the rate of healing by 16% (P Ͻ 0.05) compared with saline-treated controls. In contrast, in the ␤2-AR Ϫ/Ϫ mice, all treatment groups demonstrated equivalent rates of wound healing. Additionally, murine corneal epithelial cell expressed the catecholamine-synthesizing enzyme tyrosine hydroxylase and detectable levels of epinephrine (184.5 pg/mg protein). CONCLUSIONS. The authors provide evidence of an endogenous autocrine catecholamine signaling pathway dependent on an intact ␤2-AR for the modulation of corneal epithelial wound repair. (Invest Ophthalmol Vis Sci

β2AR antagonists and Β2AR gene deletion both promote skin wound repair processes

Skin wound healing is a complex process requiring the coordinated, temporal orchestration of numerous cell types and biological processes to regenerate damaged tissue. Previous work has demonstrated that a functional β-adrenergic receptor autocrine/paracrine network exists in skin, but the role of β2-adrenergic receptor (β2AR) in wound healing is unknown. A range of in vitro (single-cell migration, immunoblotting, ELISA, enzyme immunoassay), ex vivo (rat aortic ring assay), and in vivo (chick chorioallantoic membrane assay, zebrafish, murine wild-type, and β2AR knockout excisional skin wound models) models were used to demonstrate that blockade or loss of β2AR gene deletion promoted wound repair, a finding that is, to our knowledge, previously unreported. Compared with vehicle-only controls, β2AR antagonism increased angiogenesis, dermal fibroblast function, and re-epithelialization, but had no effect on wound inflammation in vivo. Skin wounds in β2AR knockout mice contracted and re-epithelialized faster in the first few days of wound repair in vivo. β2AR antagonism enhanced cell motility through distinct intracellular signalling mechanisms and increased vascular endothelial growth factor secretion from keratinocytes. β2AR antagonism promoted wound repair processes in the early stages of wound repair, revealing a possible new avenue for therapeutic intervention

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

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