Purpose: Transforming growth factor (TGF-β) has a critical role in wound healing. Since hypoxia is a common feature of chronic wounds and may regulate scarring, we investigated the differential effects of hypoxia/reoxygenation on the TGF-β signaling pathway in vitro using primary human skin fibroblasts. Given the anecdotal observation that the nipple areolar complex (NAC) scars less than the adjacent breast, we set out to show biochemical differences between fibroblasts from the NAC and the adjacent breast tissue.
Methods and Materials: Primary skin fibroblasts were prepared from the NAC and adjacent breast and cultured as monolayers from breast reduction specimen. Monolayers were then exposed hypoxia for either 2 or 24 hours and then re-oxygenation for 2 hours. Various components of the TGF-β signaling cascade were analyzed using western blot analysis.
Results: Preliminary results show 2 h hypoxia upregulates and 24 h hypoxia downregulates active TGF-β with little effect on total TGF-β in human skin fibroblasts. Total Smad2 and Smad3 levels were lower in NAC cells at all time points. Total Smad2 and Smad3 levels showed an increase after 2 hours of hypoxia. 2 h of hypoxia upregulated phosphorylated Smad2, Smad3 in human skin fibroblasts while 24 h of hypoxia downregulated phosphorylated Smad3 with little effect on phosphorylated Smad2, total Smad2, or total Smad3. TGF-β RII levels were shown to be decreased after 2h of hypoxia in NAC fibroblasts but shown to be increased in breast fibroblasts. Reoxygenation (2 h) counteracted the hypoxic effect at all time points tested in human skin fibroblasts.
Conclusions: Our results demonstrate that hypoxia exerts differential effects on the components of the TGF-ƒ" signaling pathway which is critically dependent on the duration of exposure, emphasizing the dynamic and context dependent regulation of TGF-ƒ" action in skin cells. As well, preliminary results suggest that NAC and breast fibroblasts are different with respect to levels of TGF-b RII and total Smad2/Smad3. These biochemical differences may contribute to the observed anecdotal difference in scarring.