Integrin-blocking antibodies delay keratinocyte re-epithelialization in a human three-dimensional wound healing model - PubMed (original) (raw)
Integrin-blocking antibodies delay keratinocyte re-epithelialization in a human three-dimensional wound healing model
Christophe Egles et al. PLoS One. 2010.
Abstract
The alpha6beta4 integrin plays a significant role in tumor growth, angiogenesis and metastasis through modulation of growth factor signaling, and is a potentially important therapeutic target. However, alpha6beta4-mediated cell-matrix adhesion is critical in normal keratinocyte attachment, signaling and anchorage to the basement membrane through its interaction with laminin-5, raising potential risks for targeted therapy. Bioengineered Human Skin Equivalent (HSE), which have been shown to mimic their normal and wounded counterparts, have been used here to investigate the consequences of targeting beta4 to establish toxic effects on normal tissue homeostasis and epithelial wound repair. We tested two antibodies directed to different beta4 epitopes, one adhesion-blocking (ASC-8) and one non-adhesion blocking (ASC-3), and determined that these antibodies were appropriately localized to the basal surface of keratinocytes at the basement membrane interface where beta4 is expressed. While normal tissue architecture was not altered, ASC-8 induced a sub-basal split at the basement membrane in non-wounded tissue. In addition, wound closure was significantly inhibited by ASC-8, but not by ASC-3, as the epithelial tongue only covered 40 percent of the wound area at 120 hours post-wounding. These results demonstrate beta4 adhesion-blocking antibodies may have adverse effects on normal tissue, whereas antibodies directed to other epitopes may provide safer alternatives for therapy. Taken together, we conclude that these three-dimensional tissue models provide a biologically relevant platform to identify toxic effects induced by candidate therapeutics, which will allow generation of findings that are more predictive of in vivo responses early in the drug development process.
Conflict of interest statement
Competing Interests: Some of the authors of this manuscript (HAH, SC, KRM) are affiliated with Biogen-Idec, one of the funders of this study. The work described in this manuscript represents a proof of concept of a technique (or of its risks). There are no plans to patent the methodology or to protect any of the information contained in this article. This situation does not alter the authors' adherence to all the Plos ONE policies on sharing data and materials.
Figures
Figure 1. Characterization of β4-specific antibody effects on human keratinocytes.
The binding of control or β4-specific antibodies ASC-3 and ASC-8 to normal human keratinocytes (A) and HaCaT cells (B) was measured by flow cytometry. To determine the effects on adhesion, serum starved cells were either left untreated (NT), or incubated with control or β4-specific antibodies prior to plating on rat laminin-5 (C) and a mixture of human laminins (D) Relative adhesion was measured using a luciferase based luminescent viability assay. Experiments were performed twice in triplicate and results shown are from one representative experiment.
Figure 2. Immunoblocking of integrins decreases the rate of cell motility.
The initial scratch wound was carried out on uncoated culture dishes with confluent keratinocyte cultures (A), and on keratinocyte cultures treated with ASC-3 (B) and ASC-8 (C). Panels D–F illustrate repopulation of the wound surface after 24 hours by keratinocytes without the presence of antibodies (D), or in the presence of ASC-3 (E), or ASC-8 (F). Cells that migrated into the wound gap were calculated for each condition and the differences were expressed as a percentage of wound closure of NHK cell culture (G). Each bar represents the mean and the standard deviation of triplicate determinations in two separate experiments.
Figure 3. Immunodetection of the blocking antibodies in the Human Skin Equivalents.
H and E staining of tissues revealed no differences in tissue architecture or numbers of cell layers between the control (A) and the ASC-3 (B) or ASC-8 (C) treated HSEs. No antibody was detected in the control tissues not exposed to antibodies. (D). While both ASC-3 (E) and ASC-8 (F) antibodies detected inside the tissue at the junction between dermis and epidermis, indicating specific localization by target along the basement membrane interface (C and F) ASC-8 is associated with a large disruption of the tissue integrity between the dermis and epidermis (F, white arrow).
Figure 4. Immunofluorescent detection of the Basement Membrane Proteins.
The immunodetection of alpha6 integrin shows no difference between the control (A) and the presence of ASC-3 and ASC-8 antibodies in the medium (B and C). The same observation can be made for laminin-5 (D, E and F). Note that in all cases (C),(F) ASC-8 is associated with large disruption of the tissue between dermis and epidermis (white arrow).
Figure 5. Hematoxylin and Eosin staining of in vitro wound after 48 and 120 hours.
Compared to control wounds at 48 h (A) or 120 h (B) after wounding, with antibody ASC-3, epithelial tongue has covered more than 90% at 48 hour (between arrows) (C) and is totally close after 120 hours (D). However, ASC-8 completely inhibited wound closure (E). Even after 120 hours no wound closure can be observed (F). (E) Percentage of wound closure (grey bars: closure after 48 hours; black bars: closure after 120 hours). Results from two separate experiments are presented as mean±SD. Bar: 500 µm.
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