A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. ABSTRACT Catecholamines are present in saliva, but their influence on oral epithelium is not understood. Because psychological stress increases salivary catecholamines and impairs oral mucosal wound healing, we sought to determine if epithelial adrenergic signaling could link these two findings. We found that cultured human oral keratinocytes (HOK) express the α 2B -and β 2 -adrenergic receptors (ARs). Exposure of HOK to either epinephrine or the β-AR agonist, isoproterenol, reduced migratory speed and decreased in vitro scratch wound healing. Incubation with the β-AR antagonist timolol reversed the catecholamine-induced effects, indicating that the observed response is mediated by β-AR. Epinephrine treatment decreased phosphorylation of the mitogen-activated protein kinases (MAPK) ERK1/2 and p38; these decreases were also reversed with timolol. Cultured HOK express enzymes of the epinephrine synthetic pathway, and generate epinephrine. These findings demonstrate that stress-induced elevations of salivary catecholamines signal through MAPK pathways, and result in impaired oral keratinocyte migration required for healing.
Keratinocyte migration into skin wounds is the step of the healing process that correlates with the wound closure rate. Keratinocyte migration, and wound epithelialization are decreased when beta 2-adrenergic receptors (B2AR) are activated by 1 μM epinephrine/adrenaline, resulting in delayed wound healing in human and mouse skin. In the present study, we found paradoxically, that in a subset of keratinocyte strains exposure to low concentrations of epinephrine (0.1 nM) increased, rather than decreased, their migratory rate. We find that both the alpha- and the beta-adrenergic receptors are expressed in human keratinocytes, and expression of alpha-2 AR subtypes demonstrated for the first time. Therefore, we tested if the alpha-AR could be modulating the increased migratory response observed in these cell strains. By using specific inhibitors to alpha-AR, we demonstrated that blocking A2B-AR could reverse the rapid cell migration induced by the 0.1 nM epinephrine. Phosphorylation of ERK was elevated after 1–10 minutes of the low epinephrine treatment and the A2B-AR inhibitor blocked the ERK phosphorylation. The results suggest that both the A2B-AR and B2AR mediate keratinocyte migration, in which with a low level of epinephrine treatment, A2B-AR could alter the B2AR signals and regulate the migration rate.
Chronic venous leg ulcers (VLUs) are an increasingly prevalent unmet clinical challenge. Despite their high associated morbidity and mortality, the bioengineered bilayered living cellular construct (BLCC) remains the only VLU therapy FDA-approved for efficacy in 15 years, yet its healing-promoting mechanisms are not well-understood. We hypothesized that signals of the BLCC reverse the non-healing into healing-like phenotype by targeting cellular processes recognized as hallmarks of healing impairment in VLUs: unresolved chronic inflammation and fibrosis. To dissect the BLCC's in vivo mechanisms of action, we conducted a prospective randomized controlled clinical study (ClinicalTrials.gov NCT01327937) in which patients with non-healing VLUs were assigned to receive standard-care compression therapy (n¼8) or standard care plus a single BLCC application (n¼11). Biopsies were obtained from two distinct locations (the wound edge and ulcer bed) at baseline and 1 week after treatment. Specimens underwent transcriptome profiling followed by comprehensive bioinformatics pathway analysis and in vivo validation through QPCR, Western blotting, and immunofluorescence of prospectively collected VLUs. We found that the BLCC modulated key inflammatory and growth factor signals at the VLU edge (IL1B, CXCLs, and many others), orchestrating a dramatic shift in the ulcer microenvironment which reinstated immune features of the acute wound phenotype to convert it to a healing milieu. We further identified a distinct role for the BLCC in remodeling the non-healing VLU matrix, through coordinated induction of metallothioneins and MMP8, to reverse the fibrosis of the ulcer bed. Taken together, the BLCC provided integral activation and remodeling signals to chronic VLU keratinocytes and fibroblasts to promote successful wound closure.
Murine keratinocyte culture from neonatal skin is an important tool for studying the functional role of specific genes in epithelial biology. However, when the transgenic animal is only available in a geographically distant local, obtaining viable keratinocytes can be problematic. A method for transferring the isolated murine skin from collaborating labs could decrease the cost of shipping live animals, and would allow the efficient use of the tissues from the transgenic animals. Here we optimized shipping conditions and characterized the cells retrieved and cultured from mouse skin shipped for 48 h at 0 °C. The cultured keratinocytes from the control, non-shipped skin and the 2-day shipped skin were 43.6 +/- 7.8% viable, doubled every 2 days, and expressed comparable amounts of heat shock proteins and CD29/integrin beta-1. However, under the same shipping conditions, the 3-day shipped tissue failed to establish colonies in the culture. Therefore, this 2-day shipping technique allows the transfer mouse skin from distant locations with recovery of viable, propagatable keratinocytes, facilitating long-distance collaborations.
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