The mechanisms underlying fibroblast apoptosis regulated by growth factors during wound healing.

While investigating the mechanisms underlying cell death during wound healing processes, we uncovered the pro-apoptotic effects of basic fibroblast growth factor (bFGF) on granulation tissue fibroblasts following pretreatment with transforming growth factor (TGF)-beta1 in vitro. bFGF induced caspase-3 activation and apoptosis in TGF-beta1-pretreated granulation tissue-derived fibroblasts (GF-1) following bFGF treatment for 48 and 96 h. In contrast, fibroblasts that had been treated in the same manner and that originated from the uninjured dermis did not display apoptosis, indicating that the mechanisms underlying apoptosis events in fibroblasts that originate from normal dermal and wound tissues differ. In this process, we also found that bFGF inhibited Akt phosphorylation at serine 473 and induced a rapid loss of phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 in pretreated GF-1 cells, an event that coincided with the dissociation of phosphorylated FAK from the focal adhesions. Therefore, inhibition of survival signals relayed via the disrupted focal adhesion structures and inactivated Akt following bFGF treatment may lead to apoptosis in GF-1 cells pretreated with TGF-beta1. Pretreatment of GF-1 with TGF-beta1 followed by the addition of bFGF resulted in significantly greater inhibition of phosphorylation of Akt and FAK compared to treatment with TGF-beta1 or bFGF alone. The combinatorial treatment also led to proteolysis of FAK and inhibition of FAK and Akt protein expression in GF-1 cells. These findings demonstrated a significant role for the two cytokines in apoptosis of granulation tissue fibroblasts during wound healing. In vivo studies also confirmed a marked decline in phosphorylation and protein expression of Akt and FAK in bFGF-injected skin wounds. These results led to the hypothesis that temporal activation of TGF-beta1 and bFGF at the injury site promotes apoptosis in granulation tissue fibroblasts, an event that is critical for the termination of proliferative granulation tissue formation.

Basic fibroblast growth factor induces down-regulation of alpha-smooth muscle actin and reduction of myofibroblast areas in open skin wounds.

To examine the effects of basic fibroblast growth factor (bFGF) on the inhibition of alpha-smooth muscle actin (alpha-SMA) expression in dermal fibroblasts, we have established two dermal myofibroblastic cell lines positive for alpha-SMA (rat myofibroblasts [RMF] and rat myofibroblast-like [RMFL] cells) and one fibroblastic cell line negative for alpha-SMA (rat fibroblasts cells) as a model of fibroblast differentiation. In contrast to the increased expression of alpha-SMA in RMF and RMFL cells, irrespective of transforming growth factor-beta1 treatment, bFGF induced a decrease in alpha-SMA expression in the myofibroblastic cells and the reduced expression patterns of alpha-SMA differed between cells, as demonstrated by Western blot and reverse transcription polymerase chain reaction analyses. Along with the inhibition of alpha-SMA expression by bFGF, the RMF and RMFL cells also showed different activated expression of extracellular signal-regulated kinase 1/2, suggesting the involvement of extracellular signal-regulated kinase 1/2 activation in the down-regulation of alpha-SMA expression in myofibroblasts. Furthermore, an in vivo study demonstrated that bFGF administration markedly decreases the area that is positive for alpha-SMA expression in the treated wounds after day 18. In contrast, bFGF administration significantly increased the number of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) staining and alpha-SMA-positive cells at days 10 and 14, and reduced the double-positive cells rapidly after day 18. Collectively, the current investigation identified bFGF as a potent stimulator for the reduction of the myofibroblastic area in vivo, presumably because of its effects on the down-regulation of alpha-SMA expression as well as rapid induction of apoptosis in myofibroblasts.

Promoted activation of matrix metalloproteinase (MMP)-2 in keloid fibroblasts and increased expression of MMP-2 in collagen bundle regions: implications for mechanisms of keloid progression.

AIMS: Keloid is characterized by excessive deposition of collagen, resulting from aberrant extracellular matrix (ECM) production and degradation. The aim was to investigate the role of matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) in pathological wound healing in keloids. METHODS AND RESULTS: Semiquantitative analysis of 60 keloid tissue samples and 25 mature scar tissue samples demonstrated significantly increased expression of MMP-2, TIMP-2 and TIMP-3 in keloids compared with mature scars. Within keloid regions, MMP-2 expression was significantly higher in collagen bundle regions than in non-collagen bundle regions. Double immunofluorescence revealed that keloid fibroblasts between collagen bundles exhibited MMP-2, TIMP-2 and membrane-type 1 MMP (MT1-MMP) co-expression, whereas only MMP-2 expression was evident on the edge of collagen bundles. Western blot analysis and gelatin zymography of 13 keloid-derived fibroblasts (KFbs) and six normal skin dermal-derived fibroblasts (NFbs) demonstrated that unstimulated KFbs exhibited significantly increased MMP-2 activity and expression compared with NFbs under the same conditions. CONCLUSIONS: These results together indicate that MMP-2 activity can be promoted in keloid fibroblasts between collagen bundles in cooperation with TIMP-2 and MT1-MMP. This could contribute to remodelling of collagen bundle regions and invasion of fibroblasts into peripheral normal regions through promoted degradation of ECM.

Basic fibroblast growth factor in an artificial dermis promotes apoptosis and inhibits expression of alpha-smooth muscle actin, leading to reduction of wound contraction.

To clarify the mechanisms underlying declines in wound contraction caused by basic fibroblast growth factor (bFGF) and the role of autologous fibroblasts in modulating wound healing, we have examined the expression of alpha-smooth muscle actin (alpha-SMA) and apoptosis in a model of wound healing using collagen sponges with and without bFGF (1 microg) and/or fibroblasts (1 x 10(6) cells/cm(2)) applied to experimentally produced full-thickness skin wounds in rats (n=10 for each group). At 7 days postoperatively, wounds filled with a fibroblast-seeded collagen sponge (fibroblast-seeded group) displayed a greater area of collagen sponge and a smaller area of fibroblasts compared with control wounds filled with collagen sponge alone (control group). Therefore, seeding of fibroblasts in the dermal substitute might retard degradation of the collagen sponge, inhibiting fibroblast infiltration into the substitute. By day 14, wounds filled with bFGF-treated collagen sponge without fibroblast seeding (bFGF group) displayed decreased alpha-SMA expression and significantly increased apoptosis compared with other wounds. Double staining revealed that apoptosis in alpha-SMA-positive fibroblastic cells was significantly increased in the bFGF group, suggesting that bFGF treatment is a potent stimulator of myofibroblast apoptosis. Furthermore, morphometric analysis demonstrated the significant decrease in the level of wound contraction and the degree of mature collagen bundle formation in the bFGF group by day 42. The bFGF group also showed increased bFGF expression in macrophages by day 28. These results suggest that bFGF administration to an artificial dermis promotes apoptosis of alpha-SMA-positive fibroblastic cells and inhibits alpha-SMA expression in the treated wound, thus reducing wound contraction.