Response of fibroblasts to transforming growth factor-β1 on two-dimensional and in three-dimensional hyaluronan hydrogels.
Transforming growth factor-β1 (TGF-β1), an important cytokine with multiple functions, is secreted during wound healing. Previous studies have utilized two-dimensional (2D) cell culture to elucidate the functions of TGF-β1; however, 2D culture does not represent the complex three-dimensional (3D) in vivo environment. Using a synthetic hyaluronan (HA) extracellular matrix (ECM) hydrogel, we investigated the effect of TGF-β1 on fibroblasts cultured in three conditions--on tissue culture polystyrene (TCP), on HA (2D), and in HA (3D). After TGF-β1 treatment (0.1 to 20 ng/mL), morphological features and ECM regulation were analyzed by immunocytochemistry, Western blot, quantitative polymerase chain reaction, and zymogram assays. On TCP, cells showed the typical spindle shape with strong alpha smooth muscle actin (α-SMA) staining of cytoplasmic myofilaments along the cell axes after TGF-β1 treatment; on HA (2D), spindle-shape cells showed little α-SMA staining; in HA (3D), cells were smaller and rounded with less α-SMA deposition. The α-SMA gene and protein expression on TCP were significantly upregulated by TGF-β1, but TGF-β1 did not induce α-SMA expression in the presence of HA (both 2D and 3D). 3D HA culture significantly downregulated collagen I, III, and fibronectin expression, increased matrix metalloproteinase 1 and 2 (MMP1/MMP2) activity, upregulated MMP1 mRNA and downregulated TIMP3 mRNA expression. This study suggested that exogenous HA, particularly in 3D culture, appears to suppress ECM production, enhances ECM degradation and remodeling, and inhibits myofibroblast differentiation without decreasing TGF-β receptor expression.
Division of Otolaryngology-Head and Neck Surgery, Department of Surgery, University of Wisconsin Madison, Madison, Wisconsin, USA.
Xia
X
Chen
Susan L
SL
Thibeault
D000199:Actins / Q000096:biosynthesis / Q000235:genetics; D018929:Cell Culture Techniques / Q000379:methods*; D002455:Cell Division / Q000187:drug effects; D005109:Extracellular Matrix / Q000378:metabolism; D016326:Extracellular Matrix Proteins / Q000096:biosynthesis / Q000235:genetics; D005347:Fibroblasts / Q000166:cytology / Q000187:drug effects*; D005780:Gelatin / Q000737:chemistry*; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D006820:Hyaluronic Acid / Q000031:analogs & derivatives* / Q000737:chemistry*; D020100:Hydrogels / Q000737:chemistry; D020782:Matrix Metalloproteinases / Q000096:biosynthesis / Q000235:genetics; D024510:Muscle Development / Q000187:drug effects; D058628:Myofibroblasts / Q000166:cytology / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D018125:Receptors, Transforming Growth Factor beta / Q000096:biosynthesis / Q000235:genetics; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D014827:Vocal Cords / Q000166:cytology; D055815:Young Adult
C541116:ACTA2 protein, human; D000199:Actins; D016326:Extracellular Matrix Proteins; D020100:Hydrogels; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D053773:Transforming Growth Factor beta1; C513409:carbylan-GSX; D005780:Gelatin; D006820:Hyaluronic Acid; D020782:Matrix Metalloproteinases