Appearance of myofibroblasts in transforming growth factor-β1 (tgf-β1)-exposed cardiac fibroblasts in culture

There is some controversy about the presence of α-smooth muscle actin (α-SMA), i.e. myofibroblasts, in non-treated cultures of fibroblasts. In some fibroblast cultures there is no actin all while others contain α-SMA. Actin is also absent in quiescent cultures. α-SMA, desmin and angiotensin converting enzyme (ACE) protein are determined in cardiac fibroblasts cultured for 7 days in the presence or absence of TGF-β1 (10 ng/ml) by Western blotting using specific antibodies and quantified by densitometric analysis. Western blotting shows that adult (male, Wistar) fibroblasts in second passage seeded at a density of 2,600 cell/cm2 contain a small amount of ACE which is increased 6.9-fold by TGF-β1 (10 ng/ml). Control cultures of fibroblasts also contain α-SMA and TGF-β1 induces a 6.8-fold increase in this cytoskeleton protein. However, these cultures do not contain desmin, a marker of vascular smooth muscle cells. Phase contrast light microscopy also shows that TGF-β1 induces an increase in the size of fibroblasts in culture. After 7 days of incubation the control cultures of fibroblasts are confluent or even over-confluent, but the TGF-β1-treated cultures are still non-confluent. In confluent cultures the cells are smaller, round and less variable. After harvesting the fibroblasts with trypsin, they have a round shape. TGF-β1 induces an increase in the diameter and the volume of fibroblasts harvested with trypsin (1.5- and 1.85-fold respectively). Control cultures of fibroblasts contain 0.3 ± 0.04 mg protein per 106 cells and TGF-β1 induces a 2.3-fold increase in the protein content per cell. Our data show the presence of α-SMA in control cultures indicating that our fibroblast cultures are heterogenous and contain also myofibroblasts and that TGF-β1 induces a 6.9-fold increase in the quantity of α-SMA. These myofibroblasts originating from fibroblasts cultured in the presence of TGF-β1 are larger than the original fibroblasts and contain more cell protein.