Molecular markers in patients with chronic wounds to guide surgical debridement

Abstract

Chronic wounds, such as venous ulcers, are characterized by physiological impairments manifested by delays in healing, resulting in severe morbidity. Surgical debridement is routinely performed on chronic wounds because it stimulates healing. However, procedures are repeated many times on the same patient because, in contrast to tumor excision, there are no objective biological/molecular markers to guide the extent of debridement. To develop bioassays that can potentially guide surgical debridement, we assessed the pathogenesis of the patients' wound tissue before and after wound debridement. We obtained biopsies from three patients at two locations, the nonhealing edge (prior to debridement) and the adjacent, nonulcerated skin of the venous ulcers (post debridement), and evaluated their histology, biological response to wounding (migration) and gene expression profile. We found that biopsies from the nonhealing edges exhibit distinct pathogenic morphology (hyperproliferative/hyperkeratotic epidermis; dermal fibrosis; increased procollagen synthesis). Fibroblasts deriving from this location exhibit impaired migration in comparison to the cells from adjacent nonulcerated biopsies, which exhibit normalization of morphology and normal migration capacity. The nonhealing edges have a specific, identifiable, and reproducible gene expression profile. The adjacent nonulcerated biopsies have their own distinctive reproducible gene expression profile, signifying that particular wound areas can be identified by gene expression profiling. We conclude that chronic ulcers contain distinct subpopulations of cells with different capacity to heal and that gene expression profiling can be utilized to identify them. In the future, molecular markers will be developed to identify the nonimpaired tissue, thereby making surgical debridement more accurate and more efficacious.

Figure 1

Distinct wound locations have specific histology. (A) A typical venous stasis ulcer is shown. Arrows point to the regions from which biopsies were obtained: A, nonhealing edge and B, adjacent, nonulcerated skin. (B) H&E stained biopsies showing epidermis from nonhealing edge (location A); adjacent, nonulcerated skin (location B); (C) H&E stained biopsies showing dermis from nonhealing edge (location A); adjacent, nonulcerated skin (location B). Normal skin is shown on the right panel. (D) Staining with procollagen shows increased intracellular staining of the nonhealing edge (location A) when compared with adjacent, nonulcerated skin (location B). Circles demarcate the location from which enlarged images are shown (insets below). Normal skin is shown on the right panels. Scale bar: 100 μm

Figure 2

Fibroblasts grown from different wound locations exert specific phenotype. Fibroblasts deriving from Location A (top panel) exhibit characteristic phenotype (misshaped, larger in size, and clumped together). In contrast, fibroblasts deriving from Location B (middle panel) exhibit phenotype similar to normal fibroblasts (bottom panel).

Figure 3

Fibroblasts grown from distinct wound locations have different response to wounding and different migration capacity in the wound scratch essay in vitro. Fibroblasts from Location B migrate similar to normal whereas fibroblasts from the location A have diminished migration capacity. (A) Actual experiment is shown. Full lines indicate initial wound area; dotted lines demarcate migrating front of cells. (B) Histograms indicate the average coverage of scratch wounds widths in % relative to baseline wound width at the 0, 4, 8, and 24 h.

Figure 4

Distinct wound locations have specific recognizable gene expression pattern. Gene tree (a graphic presentation in which samples are grouped based on the similarity of gene expression profiles) showing different gene expression patterns between two different wound locations: Location A, nonhealing edges (left) and adjacent, nonulcerated area, Location B (right). Highly expressed genes are shown as red lines, low expressed genes are represented as green, whereas yellow color indicated intermediately expressed genes. Asterisk marks the biopsy originating from what presumably was Location B, but based on expression pattern reveals the Location A specific profile.

Figure 5

Histology analyses of incompletely derided location confirm its gene expression pattern. (A) Gene tree indicates incomplete debridement of patient 3 (*). (B) H&E staining of Location A (top panels) and Location B* (bottom panels) from Patient 3 are shown. Stratum corneum remains hyper and parakeratotic in Location B* although less prominently. Arrows point to nuclei present in cornified layer.

Figure 6

EGFR expression pattern indicate differential expression between two wound locations. EGFR is membranous in control, unwounded skin. Its expression is markedly reduced and cytoplasmic in Location A. It is more prominent in the Location B resuming membranous pattern of expression. BM = basement membrane.