Re-epithelialization of a skin wound by the migration of keratinocytes from the wound margin as an important and necessary component of wound healing. This proposal forwards the overall hypothesis that endogenous electric fields, generated by the wounded skin, provide an early cue for directed keratinocyte migration. The proposal builds on the investigators' previous finding of cathodally-directed migration (galvanotaxis) of keratinocytes in electric fields equivalent to those measured in mammalian skin wounds. The hypothesis that will be tested is that an electric field results in the lateral electrophoresis of receptor glycoproteins within the plane of the cell membrane, initiating asymmetrical signaling events which culminate in directed cellular migration. Asymmetrical calcium influx is hypothesized to be a part of this signaling pathway. The overall aim of the study is to define the mechanism of human keratinocyte galvanotaxis.
Specific aims will investigate 1) possible plasma membrane targets the EGF receptor, the urokinase PA receptor, and integrins, 2) modulation of Ca 2+ influx 3) downstream signaling kinase pathways 4) effects of keratinocyte differentiation on galvanotaxis 5) the electric field parameters optimal for galvanotaxis. For these studies, 3 models of keratinocyte migration will be utilized, each offering a distinct advantage. 1) Real-time image analysis of migrating single cells provides the ability to quantitate cell velocity, net translocation and directedness of cell travel. 2) A wounded monolayer migration provides data on how keratinocytes move at the edge of wounded contiguous sheet, akin to normal epidermis. 3) A wounded """"""""skin equivalent"""""""", or composite culture system with fibroblasts incorporated into collagen gel overlaid with a stratified cultured keratinocytes, provides an opportunity to examine re-epithelialization in the context of a 3-dimensional matrix and with cellular 'crosstalk' between epidermal keratinocyte and dermal fibroblast. Electrical stimulation of directed keratinocyte migration may ultimately provide a powerful therapeutic tool for enhancing wound healing, particularly in chronic, non-healing wounds.
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| Lulevich, Valentin; Yang, Hsin-ya; Isseroff, R Rivkah et al. (2010) Single cell mechanics of keratinocyte cells. Ultramicroscopy 110:1435-42 |
| Sivamani, Raja K; Porter, Scott M; Isseroff, R Rivkah (2009) An epinephrine-dependent mechanism for the control of UV-induced pigmentation. J Invest Dermatol 129:784-7 |
| Sivamani, Raja K; Pullar, Christine E; Manabat-Hidalgo, Catherine G et al. (2009) Stress-mediated increases in systemic and local epinephrine impair skin wound healing: potential new indication for beta blockers. PLoS Med 6:e12 |
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