Normal Scar Fibroblasts Research Articles

Effect of THBS1 on the Biological Function of Hypertrophic Scar Fibroblasts.

Hypertrophic scarring is a skin collagen disease that can occur following skin damage and is unlikely to heal or subside naturally. Since surgical treatment often worsens scarring, it is important to investigate the pathogenesis and prevention of hypertrophic scarring. Thrombospondin-1 (THBS1) is a matrix glycoprotein that can affect fibrosis by activating TGF-β1, which plays a key role in wound repair and tissue regeneration; therefore, we investigated the effects of THBS1 on the biological function of hypertrophic scar fibroblasts. THBS1 expression was measured in hypertrophic scars and adjacent tissues as well as normal fibroblasts, normal scar fibroblasts, and hypertrophic scar fibroblasts. In addition, THBS1 was overexpressed or silenced in hypertrophic scar fibroblasts to determine the effects of THBS1 on cell proliferation, apoptosis, and migration, as well as TGF-β1 expression. Finally, the role of THBS1 in hypertrophic scarring was confirmed in vivo using a mouse model. We found that THBS1 expression was increased in hypertrophic scar tissues and fibroblasts and promoted the growth and migration of hypertrophic scar fibroblasts as well as TGF-β1 expression. Interestingly, we found that si-THBS1 inhibited the occurrence and development of bleomycin-induced hypertrophic scars in vivo and downregulated TGF-β1 expression. Together, our findings suggest that THBS1 is abnormally expressed in hypertrophic scars and can induce the growth of hypertrophic scar fibroblasts by regulating TGF-β1. Consequently, THBS1 could be an ideal target for treating hypertrophic scarring.

Site-Specific Keloid Fibroblasts Alter the Behaviour of Normal Skin and Normal Scar Fibroblasts through Paracrine Signalling

Keloid disease (KD) is an abnormal cutaneous fibroproliferative disorder of unknown aetiopathogenesis. Keloid fibroblasts (KF) are implicated as mediators of elevated extracellular matrix deposition. Aberrant secretory behaviour by KF relative to normal skin fibroblasts (NF) may influence the disease state. To date, no previous reports exist on the ability of site-specific KF to induce fibrotic-like phenotypic changes in NF or normal scar fibroblasts (NS) by paracrine mechanisms. Therefore, the aim of this study was to investigate the influence of conditioned media from site-specific KF on the cellular and molecular behaviour of both NF and NS enabled by paracrine mechanisms. Conditioned media was collected from cultured primary fibroblasts during a proliferative log phase of growth including: NF, NS, peri-lesional keloid fibroblasts (PKF) and intra-lesional keloid fibroblasts (IKF). Conditioned media was used to grow NF, NS, PKF and IKF cells over 240 hrs. Cellular behavior was monitored through real time cell analysis (RTCA), proliferation rates and migration in a scratch wound assay. Fibrosis-associated marker expression was determined at both protein and gene level. PKF conditioned media treatment of both NF and NS elicited enhanced cell proliferation, spreading and viability as measured in real time over 240 hrs versus control conditioned media. Following PKF and IKF media treatments up to 240 hrs, both NF and NS showed significantly elevated proliferation rates (p<0.03) and migration in a scratch wound assay (p<0.04). Concomitant up-regulation of collagen I, fibronectin, α-SMA, PAI-1, TGF-β and CTGF (p<0.03) protein expression were also observed. Corresponding qRT-PCR analysis supported these findings (P<0.03). In all cases, conditioned media from growing marginal PKF elicited the strongest effects. In conclusion, primary NF and NS cells treated with PKF or IKF conditioned media exhibit enhanced expression of fibrosis-associated molecular markers and increased cellular activity as a result of keloid fibroblast-derived paracrine factors.

Collagen degradation in normal and pathological scarring: Aetiological implications

Introduction: Apoptosis of fibroblasts is a key wound healing event that limits scar tissue production, failure of which may be the basis of scarring pathology. The trigger for this process is unclear but seems to involve contraction of fibrillar collagen. We have previously shown that unlike dermal and normal scar fibroblasts, pathological scar cells fail to undergo collagen-contraction-induced apoptosis.

Decreased expression of inhibitory SMAD6 and SMAD7 in keloid scarring

Keloids are benign skin tumours occurring during wound healing in genetically predisposed patients. They are characterised by an abnormal deposition of extracellular matrix components, in particular collagen. There is evidence that transforming growth factor-beta (TGFbeta) is involved in keloid formation. SMAD proteins play a crucial role in TGFbeta signaling and in terminating the TGFbeta signal by a negative feedback loop through SMAD6 and 7. It is unclear how TGFbeta signaling is connected to the pathogenesis of keloids. Therefore, we investigated the expression of SMAD mRNA and proteins in keloids, in normal skin and in normal scars. Dermal fibroblasts were obtained from punch-biopsies of keloids, normal scars and normal skin. Cells were stimulated with TGFbeta1 and the expression of SMAD2, 3, 4, 6 and 7 mRNA was analysed by real time RT-PCR. Protein expression was determined by Western blot analysis. Our data demonstrate a decreased mRNA expression of the inhibitory SMAD6 and 7 in keloid fibroblasts as compared to normal scar (p<0.01) and normal skin fibroblasts (p<0.05). SMAD3 mRNA was found to be lower in keloids (p<0.01) and in normal scar fibroblasts (p<0.001) compared to normal skin fibroblasts. Our data showed for the first time a decreased expression of the inhibitory SMAD6 and SMAD7 in keloid fibroblasts. This could explain why TGFbeta signaling is not terminated in keloids leading to overexpression of extracellularmatrix in keloids. These data support a possible role of SMAD6 and 7 in the pathogenesis of keloids.

Autocrine transforming growth factor‐β1 activity and glycosaminoglycan synthesis by human cutaneous scar fibroblasts

Transforming growth factor-beta(1) is a well-known and potent biological response modifier that plays an important role in tissue repair and fibrosis. Among the extracellular constituents known to accumulate in fibrotic tissues, glycosaminoglycans are prominent. In this study we examined transforming growth factor-beta(1) synthesis by human dermal fibroblasts derived from both normal and fibrotic cutaneous tissues. We studied the influence of transforming growth factor-beta(1) on glycosaminoglycan synthesis and explored the role of transforming growth factor-beta(1) as an autocrine mediator of its own expression. These investigations are directed at understanding the persistence of the fibrotic phenotype in scarred skin. Transforming growth factor-beta(1) activity was measured by means of a mink lung epithelium growth inhibitory assay. Replicate explants (n = 3) of fibroblasts each derived from normal skin, normal scar, or hypertrophic scar were studied by adding exogenous transforming growth factor-beta(1) at a concentration range of 0 to 10 ng/ml. The resulting conditioned media were removed and assayed for transforming growth factor-beta(1) activity, then the cells were pulsed for an additional 24 hours with radiolabeled glycosaminoglycan precursor, [(3)H]-glucosamine, to evaluate glycosaminoglycan production. Cell-free glycosaminoglycan synthetic profiles were also developed. Transforming growth factor-beta(1) was found to cause a dose-dependent increase in glycosaminoglycan synthesis in hypertrophic scar and normal skin but not in normal scar fibroblasts in cell-mediated glycosaminoglycan synthesis; the reverse was observed in cell-free glycosaminoglycan synthesis, where transforming growth factor-beta(1) increased glycosaminoglycan synthesis in normal scar but not in normal skin or hypertrophic scar. Most endogenous transforming growth factor-beta(1) existed in latent form for normal skin cells but in active form for normal scar and hypertrophic scar fibroblasts.

Binding and Internalization of Hyaluronate by Human Cutaneous Fibroblasts

Hyaluronate is a ubiquitous component of mammalian extracellular matrix. It influences numerous cellular processes and accumulates in fibrotic connective tissue disorders. Recently, hyaluronate catabolism has assumed additional importance because of the introduction into clinical practice of therapeutic procedures which deposit high concentrations of hyaluronate directly into tissues. Relatively little is known about the local metabolism, fate, or long-term effects of either endogenous or exogenous hyaluronate at deposition sites. A capacity for degrading hyaluronate within connective tissues, presumably by fibroblasts, has been inferred but remains controversial because direct proof that human fibroblasts endocytose and degrade hyaluronate has been lacking.In the present study, fibroblasts from normal and fibrotic skin were incubated with [3H]-hyaluronate. Binding and internalization of radiolabeled substrate were then measured: Binding assays revealed a saturable, dose-dependent increase in cell surface-associated [3H]-hyaluronate which was enhanced by pretreatment with hyaluronidase. Similar binding curves were obtained for all cells tested. All the cell lines internalized hyaluronate; however, fibroblasts in confluent cultures internalized 3.5- to 4.2-fold more radioactivity per cell than did fibroblasts from corresponding subconfluent cultures (p ≤ 0.002). Normal scar fibroblasts showed greater capacity for generating hyaluronate-derived partial degradation products. This work provides clear evidence that human cutaneous fibroblasts are capable of both binding and internalizing hyaluronate, possibly as a prerequisite for degradation.