H. V. Vangapandu1, H. Strang1, A. Kaul1, L. Masri1, S. Saravanan1, H. Li1, Q. Miao1, X. Wang1, S. Keswani1, S. Izaddoost1, C. Coarfa1, S. Balaji1 1Baylor College Of Medicine,Surgery,Houston, TX, USA
Introduction: How different individuals scar from identical injuries define heterogeneity in wound healing. Since, fibroblasts are the main arbiters of fibrosis, exposure to stress under oxygen constraint and high bioenergetic demands could exacerbate differences in wound repair and scarring responses. Hence, it is conceivable that fibroblast energy metabolism may underlie why we scar differently. We hypothesize that scarring is differentially regulated by intrinsic fibroblast energy metabolism cues.
Methods: Skin and paired c-section scar tissue was obtained from abdominoplasty surgeries. Samples were classified as "low" or "high" scarring potential based on VSS(<3 vs. >6). Uninjured (normal) skin and scar fibroblasts were isolated, and metabolic parameters of high scarring(HS) vs. low scarring(LS) fibroblasts were characterized by assessing mitochondrial oxidative phosphorylation (OCR), glycolysis (ECAR) and % ATP production at resting state and under hypoxic conditions (Seahorse XF assays). Mitochondrial membrane potential (ΔΨm; JC-1) and reactive oxygen species (ROS; MitoSOX Red) were also measured. Differences in signaling pathways that mediate the metabolic processes were determined using phospho-proteome profiler array. p-values by ANOVA (n=3-4 patients/group).
Results: Both normal skin and scar fibroblasts of HS samples had higher basal OCR and ECAR (more energetic) than those of LS counterparts (p<0.01). We observed no difference in the ATP production between HS and LS normal skin fibroblasts, with equal % generated from mitochondrial respiration and glycolysis. Conversely, scar fibroblasts generated more ATP via glycolysis. HS normal and scar fibroblasts responded better to biochemical stress by showing a greater increase in spare respiratory and glycolytic reserve capacity upon FCCP/Oligomycin treatments as compared to LS fibroblasts (p<0.01). Similarly, under hypoxic conditions (to mirror the wound milieu), there was a dip in OCR in both HS and LS as anticipated, but HS showed a correlatively significant increase in glycolysis. ΔΨm was higher in normal skin fibroblasts from both HS and LS as compared to scar fibroblasts(p<0.001) but, within the normal skin fibroblasts, HS had more depolarized mitochondria compared to LS (p<0.01). Notably, in comparison to LS, mitochondrial ROS was higher in HS normal skin fibroblasts (p<0.05). p-HSP27 Ser82 expression was significantly lower (p<0.001) in HS fibroblasts than in LS, which is consistent with high ROS levels in HS, as HSP27 negatively regulates ROS levels to govern fibrotic and functional responses of cells.
Conclusion: Our data provides new biochemical insights on how mitochondrial bioenergetics could govern fibroblast responses to injury and why we heal differently, which could lead to the design of effective personalized biomarker-guided therapies.