Search

Subsequent to wounding, keratinocytes must quickly restore barrier function. In vitro wound models have served to elucidate mechanisms of epithelial closure and key roles for integrins alpha6beta4 and alpha3beta1. To extrapolate in vitro data to in vivo human tissues, we used ultrathin cryomicrotomy to simultaneously observe tissue ultrastructure and immunogold localization in unwounded skin and acute human cutaneous wounds. Localization of the beta4 integrin subunit in unwounded skin shows dominant hemidesmosomal association and minor basal keratinocyte lateral filopodic cell-cell expression. After wounding, beta4 dominantly localized to cytokeratin-rich regions (trailing edge hemidesmosomes) and minor association with lamellipodia (leading edge). beta4 colocalizes with alpha3 within filopodia juxtaposed to wound matrix, and increased concentrations of beta4 were found in cytoplasmic vesicles within basal keratinocytes of the migrating tongue. alpha3 integrin subunit dominantly localized to filopodia within basal keratinocyte lateral cell-cell interfaces in unwounded skin and both cell-cell and cell-matrix filopodic interactions in wounded skin. This study indicates that beta4 interacts with the extracellular environment through both stable and transient interactions and may be managed through a different endosomal trafficking pathway than alpha3. alpha3 integrin, despite its ability to respond to alternate ligands after wounding, does so through a single structure, the filopodia.

Epithelialization of normal acute wounds occurs by an orderly series of events whereby keratinocytes migrate, proliferate, and differentiate to restore barrier function. The keratinocytes in the epidermis of chronic ulcers fail to execute this series of events. To better understand the epithelial dynamics of chronic ulcers, we used immunohistochemistry to evaluate proliferation, differentiation, adhesion, and migration in keratinocytes along the margin of chronic ulcers from patients with diabetes mellitus. We compared these features with keratinocytes from the migrating epithelial tongues of acute incisional and excisional wounds from normal volunteers. Keratinocytes at the chronic ulcer edge are highly proliferative (Ki67 proliferation marker), have an activated phenotype (K16), do not stain for keratins involved in epidermal differentiation (K10 and K2), and show a reduced expression of LM-3A32 (uncleaved, precursor of the alpha3 chain of laminin 5), a key molecule present on migrating epithelium. In contrast, keratinocytes in normal acute wound migrating epithelium do not express the proliferation marker Ki67 but do express K10, K2, and LM-3A32. A better understanding of molecular mechanisms involved in keratinocyte migration may lead to molecular targets for therapies for impaired wound healing.

Immunohistochemistry (IHC) is a valuable tool for labeling structures in tissue samples. Quantification of immunolabeled structures using traditional approaches has proved to be difficult. Manual counts of IHC-stained structures are inherently biased, require multiple observers, and generate qualitative data. Stereological methods provide accurate quantification but are complex and labor-intensive when staining must be compared among large numbers of samples. In an effort to quickly, objectively, and reproducibly quantify cutaneous innervation in a large number of counterstained tissue sections, we developed a color subtractive-computer-assisted image analysis (CS-CAIA) system. To develop and test the CS-CAIA method, tissue sections of diabetic (db/db) mouse skin and their wild-type (db/-) littermates were stained by IHC for the neural marker PGP 9.5. The brown-red PGP 9.5 peroxidase stain was colorimetrically isolated through a scripted process of color background removal. The remaining stain was thresholded and binarized for computer determination of nerve profile counts (number of stained regions), area fraction (total area of nerve profiles per unit area of tissue), and area density (total number of nerve profiles per unit area of tissue). Using CS-CAIA, epidermal nerve profile counts, area fraction, and area density were significantly lower in db/db compared to db/- mice.