Alpha-Smooth Muscle Actin Expression Upregulates Fibroblast Contractile Activity

Abstract

To evaluate whether ␣-smooth muscle actin (␣-SMA) plays a role in fibroblast contractility, we first compared the contractile activity of rat subcutaneous fibroblasts (SCFs), expressing low levels of ␣-SMA, with that of lung fibroblasts (LFs), expressing high levels of ␣-SMA, with the use of silicone substrates of different stiffness degrees. On medium stiffness substrates the percentage of cells producing wrinkles was similar to that of ␣-SMA-positive cells in each fibroblast population. On high stiffness substrates, wrinkle production was limited to a subpopulation of LFs very positive for ␣-SMA. In a second approach, we measured the isotonic contraction of SCF-and LF-populated attached collagen lattices. SCFs exhibited 41% diameter reduction compared with 63% by LFs. TGF␤1 increased ␣-SMA expression and lattice contraction by SCFs to the levels of LFs; TGF␤-antagonizing agents reduced ␣-SMA expression and lattice contraction by LFs to the level of SCFs. Finally, 3T3 fibroblasts transiently or permanently transfected with ␣-SMA cDNA exhibited a significantly higher lattice contraction compared with wild-type 3T3 fibroblasts or to fibroblasts transfected with ␣-cardiac and ␤-or ␥-cytoplasmic actin. This took place in the absence of any change in smooth muscle or nonmuscle myosin heavy-chain expression. Our results indicate that an increased ␣-SMA expression is sufficient to enhance fibroblast contractile activity. INTRODUCTIONEarly during healing of an open wound, resident dermal fibroblasts proliferate from the wound margin and migrate into the provisional matrix composed of a fibrin clot. About 1 week after wounding, the provisional matrix is replaced by neo-formed connective tissue, known as granulation tissue, essentially composed of small vessels, extracellular matrix, and fibroblastic cells that become activated and modulate into myofibroblasts. The main feature of myofibroblasts is represented by an important contractile apparatus similar to that of smooth muscle (Gabbiani et al., 1971), and in particular by the neo-expression of ␣-smooth muscle actin (␣-SMA), the actin isoform typical of vascular smooth muscle cells (Skalli et al., 1986). Myofibroblasts are recognized to play a central role in closing the wound tissue, through their capacity to produce a strong contractile force (for review see Grinnell, 1994;Rønnov-Jessen et al., 1996;Powell et al., 1999;Serini and Gabbiani, 1999), possibly generated within stress fibers, similar to those present in cultured fibroblasts (Skalli et al., 1986;Serini and Gabbiani, 1999). Because wound contraction takes place when de novo expressed ␣-SMA is incorporated in stress fibers (Darby et al., 1990), it has been suggested that this actin isoform plays an important role in granulation tissue contraction (for review see Serini and Gabbiani, 1999). Although stress fibers have been proposed to function as contractile organelles (Burridge, 1981;Katoh et al., 1998) and their presence has been correlated with the production of isometric tension (Harris et al., 1981), at p...

Defective Myofibroblast Formation from Mesenchymal Stem Cells in the Aging Murine Heart

“…64,65 Herein, we have demonstrated that overexpression of a constitutively active T␤RI mutant improved the motility of aged cardiac fibroblasts but not their ability to contract collagen pads (Figure 4). Myofibroblast maturation and migration are the opposite spectra of T␤R-mediated responses; expression of ␣-SMA increases fibroblast contractile activity 66 and decreases fibroblast motility. 67 Clearly, overexpression of T␤RI promoted the actin cytoskeleton reorganization required for directed migration 68 and, therefore, partially rescued the functioning of aged fibroblasts.…”

Quaking, an RNA-Binding Protein, Is a Critical Regulator of Vascular Smooth Muscle Cell Phenotype

“…To identify single contractile cells, stably transduced sh-cont and sh-qkI HITA2 VSMCs or VSMCs overexpressing enhanced green fluorescent protein (EGFP), MYOCD-v1a, MYOCD-v1b, MYOCD-v3a, or MYOCD-v3b were seeded on deformable silicone substrates (stiffness 5 kPa; Excellness Biotech, Lausanne, Switzerland) 29 in M199 culture medium containing 1% FCS. Cells were seeded onto substrates coated with 10 µg/mL fibronectin isolated from human plasma.…”

Nicotine inhibits myofibroblast differentiation in human gingival fibroblasts

“…Using deformable silicone substrates and a three dimensional collagen lattice, a correlation between α-SMA expression level and fibroblast contraction force was found. In addition, fibroblasts transfected with α-SMA cDNA had increased contractile ability [Hinz et al, 2001]. …”