Title: Neonatal and Adult Dermal Fibroblasts Show Differences in Transforming Growth Factor (TGF-β) Secretion and TGF-β Type II Receptor Expression at Baseline and Under Constant Stretch Conditions
Abstract: Previous investigations in dysregulation of tissue fibrosis seen in chronic venous insufficiency (CVI) have used commercially available neonatal fibroblasts (nn-fbs) as controls. Adult fibroblasts (a-fbs) are commercially available but have not been used as controls to study venous disease, which occurs predominantly in the adult population. In an effort to investigate if transforming growth factor (TGF)-β alterations occur as a normal process in healthy aging fibroblasts, we examined TGF-β, TGF-βRI, and TGFβ-RII expression between commercial nn-fbs and a-fbs. We also attempted to determine if there were any differences in response to mechanical stress between healthy nn-fbs and a-fbs. Confluent early passage (P3-P5) nn-fbs and a-fbs were cultured. Flow cytometry by fluorescence activated cell sorting (FACS) was performed to determine basal levels of TGF-βRI-positive and TGF-βRII-positive cells and receptor density measured by mean fluorescent intensity (MFI). Basal levels of secreted TGF-β were quantified using enzyme-linked immunosorbent assay (ELISA). To mimic the increased stretch to which dermal fibroblasts are exposed to in patients with CVI, nn-fbs and a-fbs were cultured on collagen-coated Flexplates, and subjected to constant equibiaxial elongation (21%) for 24 hours using a Flexercell strain unit. Cells and media were harvested. TGF-β secretion in response to stress was determined by ELISA. No difference was noted in the number of TGF-βRI-positive and TGF-βRII-positive cells between a-fbs and nn-fbs; however, TGF-βRII density was reduced 40% as determined by MFI in a-fbs compared with nn-fbs (151 ± 25 vs 51 ± 27, n = 6; P = .03). In healthy commercial nn-fbs subjected to constant mechanical stress for 24 hours, there was no change in TGF-β secretion compared with the static (control) nn-fbs. However, there was a fourfold increase in TGF-β secretion (284 ± 133 vs 1076 ± 238 pg/mL, n = 4; P = 0.03) in a-fbs exposed to mechanical stress compared with their control. Previous studies investigating venous ulcer fibroblasts have shown alterations of TGF- β and their receptors. This study reveals that these alterations may occur as part of the normal aging process as demonstrated by baseline differences seen between nn-fbs and a-fbs. Furthermore, a-fbs should be considered for use as controls because they possess normal age-specific characteristics that may more closely reflect venous disease in the adult population.