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.
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