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Differential production of apoptosis-modulating proteins in patients with hypertrophic burn scar
R J Wassermann1, M Polo, P Smith
1Division of Plastic & Reconstructive Surgery, University of South Carolina School of Medicine, Columbia 29203, USA.
Background:
The biochemical and cellular pathways resulting in the production of proliferative scar in the thermally injured patient remain incompletely elucidated. A promising area of investigation is the phenomenon of programmed cell death and its modulation. The following study was designed to quantify differential levels of the bcl-2 protooncogene and the Fas cell surface receptor, two apoptosis-modulating proteins, in the peripheral blood mononuclear cell (PBMC) fractions of burn patients with hypertrophic scar versus those considered to have healed normally. The study also encompassed an immunohistochemical examination of fibroblasts in vitro, to identify differential levels of Fas, bcl-2, and interleukin converting enzyme (ICE).
Methods:
PBMC fractions were isolated from two matched burn patient cohorts of 10 patients each, the experimental group carrying the clinical and histopathologic diagnosis of hypertrophic burn scar. The supernatant from each mitogenically stimulated specimen was halved and subjected to the Fas/APO-1 enzyme-linked immunosorbent assay (ELISA) and the bcl-2 ELISA. Results for each assay were compared between groups by unpaired t tests. Further biopsy specimens of isolated proliferative scar were used in vitro to analyze the role of these apoptosis-modulating proteins and ICE. This immunoperoxidase technique was analyzed qualitatively.
Results:
The expression of the bcl-2 protein in the PBMC fractions of the burn patients with hypertrophic scar is significantly elevated in comparison to the control cohort (307.72 +/- 72.29 u/ml vs 31.55 +/- 6.73 u/ml; P = 0.0042). The quantitative levels of the Fas receptor did not differ significantly between the groups, respectively (0.3988 +/- 0.179 u/ml vs 0.2899 +/- 0.066 u/ml; P = 0.5787). Immunoperoxidase staining of proliferative scar fibroblasts and those from surrounding skin revealed relatively decreased levels of membrane-bound Fas and ICE. bcl-2 was not detectable in these specimens.
Conclusions:
Differential expression of the bcl-2 protooncogene and the Fas cell surface receptor in the PBMC fraction of patients with burn injuries may suggest a disequilibrium in a complex biochemical signaling mechanism mediating programmed cell death. The increased levels of bcl-2 could be responsible for delayed fibroblast apoptosis, resulting in the disruption of normal healing and subsequent hypertrophic scarring. This is confirmed by an in vitro examination of wound fibroblasts versus those from surrounding uninjured skin. This immunoperoxidase technique reveals a localized relative decrease in Fas and ICE, two apoptosis-inducing proteins, at the level of the fibroblast in the proliferative scar specimen.
Insights
Elevated bcl-2 protein in burn patients with hypertrophic scar suggests delayed fibroblast apoptosis, disrupting normal healing. This study quantifies bcl-2 and Fas levels in burn scar development.
Area of Science:
- Cellular and Molecular Biology
- Wound Healing Research
- Burn Injury Pathophysiology
Background:
- Programmed cell death (apoptosis) and its modulation are critical in wound healing.
- Understanding the role of apoptosis-modulating proteins like bcl-2 and Fas is key to elucidating scar formation mechanisms.
- The exact pathways leading to proliferative scar in burn patients are not fully understood.
Purpose of the Study:
- To quantify differential levels of bcl-2 protooncogene and Fas cell surface receptor in peripheral blood mononuclear cells (PBMCs) of burn patients with hypertrophic scar versus normal healing.
- To investigate differential expression of Fas, bcl-2, and interleukin converting enzyme (ICE) in fibroblasts in vitro.
Main Methods:
- Peripheral blood mononuclear cells (PBMCs) were isolated from burn patients with and without hypertrophic scarring.
- Enzyme-linked immunosorbent assays (ELISA) were used to quantify bcl-2 and Fas levels in PBMC supernatants.
- Immunohistochemistry (immunoperoxidase staining) was employed to analyze protein expression in isolated fibroblasts from proliferative scars.
Main Results:
- Significantly elevated bcl-2 protein expression was observed in PBMCs of patients with hypertrophic scars compared to controls (P = 0.0042).
- Quantitative levels of the Fas receptor did not differ significantly between the hypertrophic scar and control groups (P = 0.5787).
- In vitro analysis showed decreased levels of membrane-bound Fas and ICE in proliferative scar fibroblasts, with undetectable bcl-2.
Conclusions:
- Differential expression of bcl-2 and Fas in PBMCs suggests a disruption in programmed cell death signaling pathways in burn patients.
- Increased bcl-2 levels may lead to delayed fibroblast apoptosis, contributing to hypertrophic scar formation.
- Localized reduction of apoptosis-inducing proteins (Fas, ICE) in fibroblasts of proliferative scars supports this hypothesis.