Enhanced bioenergetic cellular activity with metabolic switch to aerobic glycolysis in Keloid and Folliculitis Keloidalis Nuchae

Abstract

Keloid scars and folliculitis keloidalis nuchae (FKN) are benign fibroproliferative dermal lesions of unknown aetiology and ill-defined treatment, which typically present in genetically susceptible individuals. Their pathognomonic hallmarks include local aggressive invasive behaviour plus high recurrence post-therapy. In view of this, we investigated proliferative and key parameters of bioenergetic cellular characteristics of site-specific keloid-derived fibroblasts (intra(centre)- and peri(margin)-lesional) and FKN compared to normal skin and normal flat non-hypertrophic scar fibroblasts as negative controls.The results showed statistically significant (P < 0.01) and variable growth dynamics with increased proliferation and migration in keloid fibroblasts, while FKN fibroblasts showed a significant (P < 0.001) increase in proliferation but similar migration profile to controls. A statistically significant metabolic switch towards aerobic glycolysis in the fibroblasts from the disease conditions was noted. Furthermore, an increase in basal glycolysis with a concomitant increase in the cellular maximum glycolytic capacity was also demonstrated in perilesional keloid and FKN fibroblasts (P < 0.05). Mitochondrial function parameters showed increased oxidative phosphorylation in the disease conditions (P < 0.05) indicating functional mitochondria. These findings further suggest that Keloids and FKN demonstrate a switch to a metabolic phenotype of aerobic glycolysis. Increased glycolytic flux inhibition is a potential mechanistic basis for future therapy.

Keywords: Cell bioenergetics; Folliculitis Keloidalis Nuchea; Glycolysis; Keloid; Metabolism; Scarring.

Fig. 1

Illustration of the experimental design and methodology of the study. Various cellular behavioural components of site-specific keloid and FKN scars were compared against normal skin and normal scar controls. Subsequently, differences in the bioenergetics of these cells from the different lesions were established. Skin - normal non-scarred skin; N Scar - normal flat non-hypertrophic scar; I Keloid - intralesional keloid; P Keloid - perilesional keloid, FKN - folliculitis keloidalis nuchae

Fig. 2

Summary of isolation and cell culture protocol of primary human dermal fibroblast cells. Figure created with Motifolio Toolkit (Motifolio Inc, Ellicott City, MD)

Fig. 3

Inherent fibroblast cellular proliferation profiles measured by Real Time Cell Analyzer (RTCA). An xCELLigence RTCA instrument was used to measure the proliferation of the cells by growing cells in 10% FBS growth factor at a density of 1 × 10⁴ cells per well of the RTCA plate. Growth curves were conducted over a 4-day period. Results are representative of data from 3 independent biological experiments with 2 replicates. Significance was set as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Two-way ANOVA with Tukey HSD post-hoc test

Fig. 4

Scratch motility assay determination of the migratory ability of various disease and control dermal fibroblasts. (A) 2D motility assay was used to establish the migration. A linear wound was made by scratching through the monolayer with a pipette tip after cells were confluent. Wound closure was measured over a 12 h period. (B) Quantification of average cell migration area. Keloids displayed increased migration. Results are representative of data from 3 independent biological experiments with 3 replicates. Significance was set as *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Two-way ANOVA with Tukey HSD post-hoc test

Fig. 5

General cell energy phenotype assays including measurement of the Extracellular Acidification Rate (ECAR) and Oxygen Consumption Rate (OCR) in various disease and control dermal fibroblasts. Results of Cell Energy Phenotype XF Flux analyser test for both (A) OCR (pmol/min) and (B) ECAR (mpH/min) simultaneously. Keloid and FKN fibroblasts showed increased metabolic potential. Results are representative of data from 4 independent biological experiments with 3 replicates. Significance was set as *, P < 0.05; **, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; Two-way ANOVA with Tukey HSD post-hoc test

Fig. 6

Bioenergetics parameters related to glycolysis measured by Extracellular Acidification Rate (ECAR) in various disease and control dermal fibroblasts. (A) Profiles of Glycolysis Stress XF Flux analyser measurements of ECAR (mpH/min) in disease fibroblasts relative to controls. Arrows indicate injection of glucose and the specific stressors into the media. (B-E) Values of the different parameters of mitochondrial respiration. Keloid and FKN fibroblasts exhibit augmented glycolysis. Results are representative of data from 4 independent biological experiments with 3 replicates. Significance was set as *, P < 0.05; **, P < 0.01; ***, P < 0.001; Two-way ANOVA with Tukey HSD post-hoc test

Fig. 7

Bioenergetics parameters related to respiration in mitochondria measured by Oxygen Consumption Rate (OCR) in various disease and control dermal fibroblasts. (A) Profiles of Mito Stress XF Flux analyser measurements of OCR in disease fibroblasts relative to controls. Arrows indicate injection of the specific stressors into the media. (B-H) Values of the different parameters of mitochondrial respiration. Keloid and FKN fibroblasts have functional mitochondria capable of mitochondrial respiration. Results are representative of data from 3 independent experiments with 3 replicates. Significance was set as *, P < 0.05; **, P < 0.01; ***, P < 0.001; Two-way ANOVA with Tukey HSD post-hoc test