Ghassan M. Saed

p53 and apoptosis alterations in keloids and keloid fibroblasts

Keloids are the result of a dysregulated wound‐healing process and are characterized by formation of excess scar tissue that proliferates beyond the boundaries of the inciting wound. In this study, we investigated the expression of key proteins involved in regulating apoptosis in keloids. Twenty archival paraffin‐embedded keloid samples were randomly selected for an immunoperoxidase assay with antibodies against fas, p53, bcl‐2, and bcl‐x proteins using the target antigen‐retrieval technique. Apoptosis was assessed in keloids and normal skin and in keloid and normal fibroblasts by the TdT‐mediated dUTP nick‐end labeling (tunel) assay on tissue sections, fibroblast cultures, and by flow cytometry for cell suspensions. We found that 18 of 20 keloids expressed p53 protein; bcl‐2 was expressed by keloid fibroblasts in 19 of 20 keloids, and all specimens had prominent fas expression throughout the tissue. The distribution of these three antigens was regional within each lesion and followed a consistent pattern of p53 and bcl‐2 expression colocalized to the hypercellular, peripheral areas of each keloid in a perinuclear pattern (p < .001). In contrast, an inverse distribution of fas expression was shown, with staining being more diffuse across the cell surfaces and limited to the central, more hypocellular regions in 16 of 17 keloids (p < .001). There was no specific staining pattern in these keloids with antihuman bcl‐x. In vitro studies on cultured keloid fibroblasts (derived from six patients) revealed maintenance of the p53+, bcl‐2+ phenotype up to passage 10. Neither neonatal nor normal adult skin fibroblasts expressed either antigen but could be induced to express p53 by exposure to adriamycin. Keloid lesions and keloid fibroblasts were found to have lower rates of apoptosis than normal controls. Keloid fibroblasts displayed enhanced apoptosis rates in response to hydrocortisone, γ interferon and hypoxia treatment as compared with normal adult fibroblasts. Focal dysregulation of p53 combined with upregulation of bcl‐2 may help produce a combination of increased cell proliferation and decreased cell death in the younger hypercellular areas of the keloid. This phenotype is reversed in the older areas of the keloid and may prevent malignan degeneration, thus favoring normal apoptosis as evidenced by prominent fas expression.

Cytokine mRNA Changes during the Treatment of Hypertrophic Scars with Silicone and Nonsilicone Gel Dressings

background Treatment of hypertrophic scars can be difficult for both patients and physicians. Silicone‐containing gel dressings have been reported to be an effective alternative treatment for hypertrophic scars, yet the mechanism of action of these dressings is unknown. objective To determine whether silicone is an essential factor in the treatment of hypertrophic scars and investigate the effects of occlusive dressing therapy on the expression of key wound healing mediators. methods A pilot paired comparison, nonrandomized study was conducted comparing a silicone gel sheeting (Silastic [SGS]) with a hydrogel dressing (Clear Site). The effects of the dressings were compared side by side in the treatment of 15 hypertrophic scars at both the clinical and molecular levels through the use of reverse transcriptase/polymerase chain reaction to evaluate effects on the expression of interleukin 8 (IL‐8), basic fibroblast growth factor (bFGF), granulocyte‐macrophage colony‐stimulating factor (GMCSF), epidermal growth factor (EGF), transforming growth factor beta (TGF‐&bgr;), and fibronectin. results Comparable clinical improvement of the hypertrophic scars was obtained with both dressings. Treatment of hypertrophic scars resulted in increased mean levels of IL‐8, bFGF, and GMCSF mRNA; while mean TGF&bgr; and fibronectin mRNAs decreased after treatment with both dressings. Comparison between the two dressings revealed significant changes in IL‐8 and fibronectin mRNA levels after treatment with Clear Site, while only fibronectin changes were significant after treatment with SGS with respect to normal skin. Only Clear Site induced significant changes in IL‐8 and bFGF levels when untreated scars were compared with posttreatment lesions, suggesting that the hydrogel augments collagenolysis via promotion of inflammation. conclusions This study demonstrates that silicone is not a necessary component of occlusive dressings in the treatment of hypertrophic scars. The pathogenesis of hypertrophic scars is further elucidated by demonstrating that there is molecular evidence for extensive connective tissue remodeling occurring during occlusive dressing therapy.

Neuronal β-amyloid precursor protein gene expression: regulation by aurintricarboxylic acid

beta-Amyloid precursor protein (beta-APP) and its derivative, amyloid beta-protein (beta-A4), may cause death of differentiated neurons and aurintricarboxylic acid (ATA), a metabolic inhibitor, improves neuronal survival. Therefore, we studied the effect of ATA on neuronal beta-APP gene expression. ATA decreased beta-APP mRNA levels by increasing its degradation, without changing the rate of transcription. ATA decreased both steady state and interleukin-1 (IL1)-induced increase in beta-APP mRNA levels. These effects of ATA were associated with rounding of cells suggestive of decreased cell adhesion or neurite retraction that was completely reversible when ATA was removed. However, beta-APP mRNA levels continued to remain suppressed in neurons that were actively regrowing neurites following discontinuation of ATA. In studies carried out upto 24 h, ATA did not damage cells as determined by Trypan blue exclusion, lactate dehydrogenase (LDH)-release and transmission electron microscopy. The findings suggest that constitutive or steady state levels of beta-APP mRNA may not be essential for the survival and growth of neurons and that ATA suppresses beta-APP expression without causing cell damage. These observations may be a basis for studying whether ATA or a related compound could beneficially regulate beta-APP levels in vivo.