Christine E. Pullar

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

Background Stress, both acute and chronic, can impair cutaneous wound repair, which has previously been mechanistically ascribed to stress-induced elevations of cortisol. Here we aimed to examine an alternate explanation that the stress-induced hormone epinephrine directly impairs keratinocyte motility and wound re-epithelialization. Burn wounds are examined as a prototype of a high-stress, high-epinephrine, wound environment. Because keratinocytes express the β2-adrenergic receptor (β2AR), another study objective was to determine whether β2AR antagonists could block epinephrine effects on healing and improve wound repair. Methods and Findings Migratory rates of normal human keratinocytes exposed to physiologically relevant levels of epinephrine were measured. To determine the role of the receptor, keratinocytes derived from animals in which the β2AR had been genetically deleted were similarly examined. The rate of healing of burn wounds generated in excised human skin in high and low epinephrine environments was measured. We utilized an in vivo burn wound model in animals with implanted pumps to deliver β2AR active drugs to study how these alter healing in vivo. Immunocytochemistry and immunoblotting were used to examine the up-regulation of catecholamine synthetic enzymes in burned tissue, and immunoassay for epinephrine determined the levels of this catecholamine in affected tissue and in the circulation. When epinephrine levels in the culture medium are elevated to the range found in burn-stressed animals, the migratory rate of both cultured human and murine keratinocytes is impaired (reduced by 76%, 95% confidence interval [CI] 56%–95% in humans, p < 0.001, and by 36%, 95% CI 24%–49% in mice, p = 0.001), and wound re-epithelialization in explanted burned human skin is delayed (by 23%, 95% CI 10%–36%, p = 0.001), as compared to cells or tissues incubated in medium without added epinephrine. This impairment is reversed by β2AR antagonists, is absent in murine keratinocytes that are genetically depleted of the β2AR, and is reproduced by incubation of keratinocytes with other β2AR-specific agonists. Activation of the β2AR in cultured keratinocytes signals the down-regulation of the AKT pathway, accompanied by a stabilization of the actin cytoskeleton and an increase in focal adhesion formation, resulting in a nonmigratory phenotype. Burn wound injury in excised human skin also rapidly up-regulates the intra-epithelial expression of the epinephrine synthesizing enzyme phenylethanolamine-N-methyltransferase, and tissue levels of epinephrine rise dramatically (15-fold) in the burn wounded tissue (values of epinephrine expressed as pg/ug protein ± standard error of the mean: unburned control, 0.6 ± 0.36; immediately postburn, 9.6 ± 1.58; 2 h postburn, 3.1 ± 1.08; 24 h post-burn, 6.7 ± 0.94). Finally, using an animal burn wound model (20% body surface in mice), we found that systemic treatment with βAR antagonists results in a significant increase (44%, 95% CI 27%–61%, p < 0.00000001) in the rate of burn wound re-epithelialization. Conclusions This work demonstrates an alternate pathway by which stress can impair healing: by stress-induced elevation of epinephrine levels resulting in activation of the keratinocyte β2AR and the impairment of cell motility and wound re-epithelialization. Furthermore, since the burn wound locally generates epinephrine in response to wounding, epinephrine levels are locally, as well as systemically, elevated, and wound healing is impacted by these dual mechanisms. Treatment with beta adrenergic antagonists significantly improves the rate of burn wound re-epithelialization. This work suggests that specific β2AR antagonists may be apt, near-term translational therapeutic targets for enhancing burn wound healing, and may provide a novel, low-cost, safe approach to improving skin wound repair in the stressed individual.

β2-Adrenergic receptor activation delays wound healing

Keratinocytes migrate directionally into the wound bed to initiate re‐epithelialization, necessary for wound closure and restoration of barrier function. They solely express the β2‐adrenergic receptor (β2‐AR) subtype of β‐ARs and can also synthesize β‐AR agonists generating a hormonal mediator network in the skin. Emerging studies from our laboratory demonstrate that β‐AR agonists decrease keratinocyte migration via a protein phosphatase (PP) 2A‐dependent mechanism. Here we have extended our investigations to observe the effects of β2‐AR activation on keratinocyte polarization, migration, and ERK phosphorylation at the wound edge, cytoskeletal organization, phospho‐ERK intracellular localization, proliferation, human skin wound re‐epithelialization, wound‐induced ERK phosphorylation, and murine skin wound healing. We demonstrate that in keratinocytes, β2‐AR activation is antimotogenic and anti‐mitogenic with both mechanisms being PP2A dependent. β2‐AR activation dramatically alters the organization of the actin cytoskeleton and prevents localization of phospho‐ERK to the lamellipodial edge and its colocalization with vinculin. Finally, we demonstrate a β2‐AR‐mediated delay in re‐epithelialization and decrease in wound‐induced epidermal ERK phosphorylation in human skin wounds and a delay in re‐epithelialization in murine tail‐clip wounds. Our work uncovers novel keratinocyte biology and a previously unrecognized role for the adrenergic hormonal mediator network in the wound repair process.— Pullar, C. E., Grahn, J. C., Liu, W., Isseroff, R. R. β2‐Adrenergic receptor activation delays wound healing. FASEB J. 20, 76 −86 (2006)

β-Adrenergic Receptor Antagonists Accelerate Skin Wound Healing EVIDENCE FOR A CATECHOLAMINE SYNTHESIS NETWORK IN THE EPIDERMIS

The skin is our primary defense against noxious environmental agents. Upon injury, keratinocytes migrate directionally into the wound bed to initiate re-epithelialization, essential for wound repair and restoration of barrier integrity. Keratinocytes express a high level of β2-adrenergic receptors (β2-ARs) that appear to play a role in cutaneous homeostasis as aberrations in either keratinocyte β2-AR function or density are associated with various skin diseases. Here we report the novel finding that β-AR antagonists promote wound re-epithelialization in a “chronic” human skin wound-healing model. β-AR antagonists increase ERK phosphorylation, the rate of keratinocyte migration, electric field-directed migration, and ultimately accelerate human skin wound re-epithelialization. We demonstrate that keratinocytes express two key enzymes required for catecholamine (β-AR agonist) synthesis, tyrosine hydroxylase and phenylethanolamine-N-methyl transferase, both localized within keratinocyte cytoplasmic vesicles. Finally, we confirm the synthesis of epinephrine by measuring the endogenously synthesized catecholamine in keratinocyte extracts. Previously, we have demonstrated that β-AR agonists delay wound re-epithelialization. Here we report that the mechanism for the β-AR antagonist-mediated augmentation of wound repair is due to β2-AR blockade, preventing the binding of endogenously synthesized epinephrine. Our work describes an endogenous β-AR mediator network in the skin that can temporally regulate skin wound repair. Further investigation of this network will improve our understanding of both the skin repair process and the multiple modes of action of one of the most frequently prescribed class of drugs, hopefully resulting in a new treatment for chronic wounds.

Interaction of Phosphorylated FcϵRIγ Immunoglobulin Receptor Tyrosine Activation Motif-based Peptides with Dual and Single SH2 Domains of p72syk ASSESSMENT OF BINDING PARAMETERS AND REAL TIME BINDING KINETICS

To examine the characteristics of the interaction of the FcεRIγ ITAM with the SH2 domains of p72syk, the binding of an 125I-labeled dual phosphorylated FcεRIγ ITAM-based peptide to the p72syk SH2 domains was monitored utilizing a novel scintillation proximity based assay. The Kd for this interaction, determined from the saturation binding isotherm, was 1.4 nM. This high affinity binding was reflected in the rapid rate of association for the peptide binding to the SH2 domains. Competition studies utilizing a soluble C-terminal SH2 domain knockout and N-terminal SH2 domain knockouts revealed that both domains contribute cooperatively to the high affinity binding. Unlabeled dual phosphorylated peptide competed with the 125I-labeled peptide for binding to the dual p72syk SH2 domains with an IC50 value of 4.8 nM. Monophosphorylated 24-mer FcεRIγ ITAM peptides, and phosphotyrosine also competed for binding, but with substantially higher IC50 values. This, and other data discussed, suggest that high affinity binding requires both tyrosine residues to be phosphorylated and that the preferred binding orientation of the ITAM is such that the N-terminal phosphotyrosine occupies the C-terminal SH2 domain and the C-terminal phosphotyrosine occupies the N-terminal SH2 domain.

The β2-adrenergic receptor activates pro-migratory and pro-proliferative pathways in dermal fibroblasts via divergent mechanisms

Dermal fibroblasts are required for skin wound repair; they migrate into the wound bed, proliferate, synthesize extracellular matrix components and contract the wound. Although fibroblasts express β2-adrenergic receptors (β2-AR) and cutaneous keratinocytes can synthesize β-AR agonists (catecholamines), the functional significance of this hormonal mediator network in the skin has not been addressed. Emerging studies from our laboratory demonstrate that β2-AR activation modulates keratinocyte migration, essential for wound re-epithelialization. Here we describe an investigation of the effects of β2-AR activation on the dermal component of wound healing. We examined β2-AR-mediated regulation of biological processes in dermal fibroblasts that are critical for wound repair: migration, proliferation, contractile ability and cytoskeletal conformation. We provide evidence for the activation of at least two divergent β2-AR-mediated signaling pathways in dermal fibroblasts, a Src-dependent pro-migratory pathway, transduced through the epidermal growth factor receptor and extracellular signal-regulated kinase, and a PKA-dependent pro-proliferative pathway. β2-AR activation attenuates collagen gel contraction and alters the actin cytoskeleton and focal adhesion distribution through PKA-dependent mechanisms. Our work uncovers a previously unrecognized role for the adrenergic hormonal mediator network in the cutaneous wound repair process. Exploiting these divergent β2-AR agonist responses in cutaneous cells may generate novel therapeutic approaches for the control of wound healing.

Angiogenesis is induced and wound size is reduced by electrical stimulation in an acute wound healing model in human skin

Angiogenesis is critical for wound healing. Insufficient angiogenesis can result in impaired wound healing and chronic wound formation. Electrical stimulation (ES) has been shown to enhance angiogenesis. We previously showed that ES enhanced angiogenesis in acute wounds at one time point (day 14). The aim of this study was to further evaluate the role of ES in affecting angiogenesis during the acute phase of cutaneous wound healing over multiple time points. We compared the angiogenic response to wounding in 40 healthy volunteers (divided into two groups and randomised), treated with ES (post-ES) and compared them to secondary intention wound healing (control). Biopsy time points monitored were days 0, 3, 7, 10, 14. Objective non-invasive measures and H&E analysis were performed in addition to immunohistochemistry (IHC) and Western blotting (WB). Wound volume was significantly reduced on D7, 10 and 14 post-ES (p = 0.003, p = 0.002, p<0.001 respectively), surface area was reduced on days 10 (p = 0.001) and 14 (p<0.001) and wound diameter reduced on days 10 (p = 0.009) and 14 (p = 0.002). Blood flow increased significantly post-ES on D10 (p = 0.002) and 14 (p = 0.001). Angiogenic markers were up-regulated following ES application; protein analysis by IHC showed an increase (p<0.05) in VEGF-A expression by ES treatment on days 7, 10 and 14 (39%, 27% and 35% respectively) and PLGF expression on days 3 and 7 (40% on both days), compared to normal healing. Similarly, WB demonstrated an increase (p<0.05) in PLGF on days 7 and 14 (51% and 35% respectively). WB studies showed a significant increase of 30% (p>0.05) on day 14 in VEGF-A expression post-ES compared to controls. Furthermore, organisation of granulation tissue was improved on day 14 post-ES. This randomised controlled trial has shown that ES enhanced wound healing by reduced wound dimensions and increased VEGF-A and PLGF expression in acute cutaneous wounds, which further substantiates the role of ES in up-regulating angiogenesis as observed over multiple time points. This therapeutic approach may have potential application for clinical management of delayed and chronic wounds.

ß-adrenergic receptor agonists delay while antagonists accelerate epithelial wound healing: Evidence of an endogenous adrenergic network within the corneal epithelium

Wound healing is a complex and well‐orchestrated biological process. Corneal epithelial cells (CECs) must respond quickly to trauma to rapidly restore barrier function and protect the eye from noxious agents. They express a high level of ß2‐adrenergic receptors but their function is unknown. Here, we report the novel finding that they form part of a regulatory network in the corneal epithelium, capable of modulating corneal epithelial wound repair. ß‐adrenergic receptor agonists delay CEC migration via a protein phosphatase 2A‐mediated mechanism and decrease both electric field‐directed migration and corneal wound healing. Conversely, ß‐adrenergic receptor antagonists accelerate CEC migration, enhance electric field‐mediated directional migration, and promote corneal wound repair. We demonstrate that CECs express key enzymes required for epinephrine (ß‐adrenergic receptor agonist) synthesis in the cytoplasm and can detect epinephrine in cell extracts. We propose that the mechanism for the pro‐motogenic effect of the ß‐adrenergic antagonist is blockade of the ß2‐adrenergic receptor preventing autocrine catecholamine binding. Further investigation of this network will improve our understanding of one of the most frequently prescribed class of drugs. J. Cell. Physiol. 211: 261–272, 2007.

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.

Tensin 2 modulates cell contractility in 3D collagen gels through the RhoGAP DLC1

Cytoskeletal proteins of the tensin family couple integrins to the actin cytoskeleton. They are found in both focal adhesions and the fibrillar adhesions formed between cells and the fibronectin matrix. There are four tensin genes which encode three large (∼200 kDa) tensin isoforms (tensin 1, 2, 3) and one short isoform (cten). However, the subcellular localization and function of the individual isoforms is poorly understood. Using human foreskin fibroblasts (HFFs), and imaging on both fixed and live cells, we show that GFP‐tensin 2 is enriched in dynamic focal adhesions at the leading edge of the cell, whereas GFP‐tensin 3 translocates rearward, and is enriched in fibrillar adhesions. To investigate the possible role of tensins in cell‐matrix remodeling, we used siRNAs to knockdown each tensin isoform. We discovered that tensin 2 knockdown significantly reduced the ability of HFFs to contract 3D collagen gels, whilst no effect on fibronectin fibrillogenesis was observed. This inhibition of collagen gel contraction was associated with a substantial reduction in Rho activity, and it was reversed by depletion of DLC1, a RhoGAP that binds to tensin in focal adhesions. These findings suggest that focal adhesion‐localized tensin 2 negatively regulates DLC1 to permit Rho‐mediated actomyosin contraction and remodeling of collagen fibers. J. Cell. Biochem. 109: 808–817, 2010.

β-Adrenergic receptor modulation of wound repair

Adrenergic receptors and their downstream effector molecules are expressed in all cell types in the skin, and it is only recently that functionality of the catecholamine agonist activated signaling in the cutaneous repair process has been revealed. In addition to responding to systemic elevations in catecholamines (as in stress situations) or to pharmacologically administered adrenergic agonists, epidermal keratinocytes themselves can synthesize catecholamine ligands. They also respond to these systemic or self-generated agonists via receptor mediated signaling, resulting in altered migration, and changes in wound re-epithelialization. Endothelial cells, inflammatory cells, dermal fibroblasts, and mesenchymal stem cells, all cells that contribute to the wound repair process, express multiple subtypes of adrenergic receptors and exhibit responses that can be either contribute or impair healing-and occasionally, depending on the species and assay conditions, results can be conflicting. There is still much to be uncovered regarding how this self-contained autocrine and paracrine signaling system contributes to cutaneous wound repair.