RAC activity in keloid disease: comparative analysis of fibroblasts from margin of keloid to its surrounding normal skin

Abstract

Background: Keloids are characterized by excess collagen deposition within the dermis. Although the exact cause of the potentially overactive fibroblasts has yet to be elucidated, many etiological possibilities have been suggested. As fibroblasts originating from keloids appear to have an increased migration and proliferation rate, cell-signaling studies examining these factors may offer an opportunity to further our understanding of the pathogenesis of this disease. One of such cell-signaling messengers is the enzyme Ras-related C3 botulinum toxin substrate (RAC), which has never been investigated in keloid scars.

Objective: This study explores the role of RAC activity in keloid disease.

Method: Primary fibroblast cell lines were established from the margin of keloid (KF) scars as well as from the surrounding normal tissue (NF) from one anatomical site of the same patient. Migration and proliferation assays were performed, comparing matching NFs and KFs, and after cell lysis, RAC activity was assessed.

Results: Comparing fibroblasts from 3 different patients, KFs migrated (P < .05) and proliferated (P < .05) faster than NFs. The activity levels of RAC were increased in KFs compared with NFs.

Conclusion: KFs migrate and proliferate faster than NFs. RAC activity increases in KFs when compared with NFs. Inhibition of RAC could lead to a new therapeutic approach.

Figure 1

Schematic representation of the extralesional excision. Fibroblasts from the margin of the scar are entitled KF, and fibroblasts from the normal surrounding tissue, NF.

Figure 2

Fibroblast migration. The distance migrated across a gap of cells created by a pipette tip on a fully confluent cell culture. KS1, KS2, and KS3, respectively, show a significant higher migration rate after 34 hours in KFs than in NFs. After 42 hours, the difference in migration is nonsignificant, as the gap was crossed.

Figure 3

Absorbance of stained fibroblasts. Example of daily averages of absorbance readings (n = 3) of stained fixed-cell cultures. As the absorbance is directly related to the amount of cells in each well, the total number of cells present is proportional to the absorbance. Day 1 shows similar absorbance, proving that the amount of cells plated is similar between the 2 groups. On day 4, the proliferation is maximal and the KF rate of proliferation slows down compared with wild type (WT) because confluence is reached. Fibroblasts from the margin of the keloid proliferate at a faster rate than fibroblasts from the unaffected skin.

Figure 4

Ras-related C3 botulinum toxin substrate (RAC) activity Western blot. RAC activity of fibroblasts originating from normal tissue (WT) and from the margin of the keloid tissues (Margin) is depicted on these representative Western blots. Cell sample KS1 was located on the scalp of a 36-year-old man. KS2 was located on the ear of a 33-year-old woman. KS3 was resected from above the scapula of a 37-year-old woman. The Western blot bands were quantified using SynGene Gene Tools program. The intensity of each band was measured, and the ratio of RAC activity/RAC total was calculated to compare the relative activity in each sample. Fibroblasts originating from keloid margins show a higher RAC activity than fibroblasts from the normal surrounding skin (WT).