Extracorporeal Shock Wave Therapy Alters the Expression of Fibrosis-Related Molecules in Fibroblast Derived from Human Hypertrophic Scar

Abstract

Extracorporeal shock wave therapy (ESWT) considerably improves the appearance and symptoms of post-burn hypertrophic scars (HTS). However, the mechanism underlying the observed beneficial effects is not well understood. The objective of this study was to elucidate the mechanism underlying changes in cellular and molecular biology that is induced by ESWT of fibroblasts derived from scar tissue (HTSFs). We cultured primary dermal fibroblasts derived from human HTS and exposed these cells to 1000 impulses of 0.03, 0.1, and 0.3 mJ/mm². At 24 h and 72 h after treatment, real-time PCR and western blotting were used to detect mRNA and protein expression, respectively, and cell viability and mobility were assessed. While HTSF viability was not affected, migration was decreased by ESWT. Transforming growth factor beta 1 (TGF-β1) expression was reduced and alpha smooth muscle actin (α-SMA), collagen-I, fibronectin, and twist-1 were reduced significantly after ESWT. Expression of E-cadherin was increased, while that of N-cadherin was reduced. Expression of inhibitor of DNA binding 1 and 2 was increased. In conclusion, suppressed epithelial-mesenchymal transition might be responsible for the anti-scarring effect of ESWT, and has potential as a therapeutic target in the management of post-burn scars.

Keywords: burn hypertrophic scar; epithelial-mesenchymal transition; extracorporeal shock wave therapy; hypertrophic scar-derived fibroblast; inhibitor of DNA binding protein.

Figure 1

The characteristics of fibroblasts derived from scar tissue (HTSFs). Matched human normal fibroblasts (HNF) and HTSF were cultured from four patients with post burn hypertrophic scar tissue. Protein expression of transforming growth factor beta 1 (TGF-β1), alpha smooth muscle actin (α-SMA), COL-Ι (collagen type Ι), COL-III (collagen type III), FN (fibronectin), Vimentin, fibroblast specific protein 1 (FSP-1), Twist-1 and N-cad (N-cadherin) was significantly higher in HTSFs compared with HNF from skin dermis. The protein expression of E-cad (E-cadherin), inhibitor of DNA binding protein 1 (ID-1) and inhibitor of DNA binding protein 2 (ID-2) were lower in HTSFs when compared with HNF from skin dermis. That expression of those proteins was measured by western blotting against specific antibody. The intensity of band was normalized with that of loading control, β-actin or lamin B1, respectively; HNF, Human normal skin derived fibroblast; HTSF, human hypertrophic scar derived fibroblast. * p < 0.05 vs. the corresponding HNF.

Figure 2

Experimental schematic diagrams and viability of HTSFs. The dermis was separated from human hypertrophic scar tissue by dispase, and then digested with collagenase type IV. HTSF was released, collected, suspended in medium, and continue cultured. After detachment, HTSFs were suspended in to a 17 mL conical tube. ESWT is performed with 1000 impulse/cm² at 0.03, 0.1, and 0.3 mJ/mm² of energy flux densities. Then, HTSFs were seeded on 96 well cell culture plates for viability assays, μ-dish for migration assays, and T75 culture plates for RT-PCR and western blot (0 h). After 24 h, the viability of HTSF was measured. Once removes insert of μ-dish, the HTSF begins to move, and then analyzed after migration 24 and 48 h (48 and 72 h after ESWT). Real time polymerase chain reaction (RT-PCR) and western blot were performed 24 h and 72 h after plating, respectively (A). ESWT no influence on viability of HTSFs (B). Cell viability was determined using an Cell Titer-Glo^® Luminescent cell viability assay kit 24 h after ESWT. Each group was assayed in sextuplicate, and the experiments were performed at three times independently. HTSF viability was expressed as a percentage value of untreated cells. Un: untreated cells.

Figure 3

Extracorporeal shockwave therapy (ESWT) decreases the expression of TGF-β1 and alpha smooth muscle actin (α–SMA) in HTSFs. HTSF was cultured from four patients with post burn hypertrophic scar tissue. The mRNA expression of TGF-β1 (A) and α–SMA (C) were measured 24 h and 72 h after ESWT using a Light Cycler real-time PCR system. Each sample was assayed in duplicate, and experiments were performed least three times independently. The mRNA expression was normalized as ratio = 2^−∆∆Ct, and data are the mean ± S.E. * p < 0.05 and † p < 0.01 vs. the corresponding untreated control group. Protein expression of TGF-β1 (B) and α–SMA (D) were measured with western blot analysis 24 and 72 h after ESWT, respectively. The protein expression was normalized with β-actin, respectively; and data are the mean ± S.E. * p < 0.05 vs. the corresponding untreated control group. Un: Untreated cells.

Figure 4

ESWT decreases the expression of extracellular matrix protein in HTSFs. HTSF was cultured from four patients with post burn hypertrophic scar tissue. The mRNA expression of collagen-I (A) and fibronectin (B) were measured 24 and 72 h after ESWT using a Light Cycler real-time PCR system. Each sample was assayed in duplicate, and experiments were performed least three times independently. The mRNA expression was normalized as ratio = 2^−∆∆Ct, and data are the mean ± S.E. * p < 0.05 and † p < 0.01 vs. the corresponding untreated control group. Protein expression of collagen-Ι (C) and fibronectin (D) were measured with western blot analysis 24 and 72 h after ESWT, respectively. The protein expression was normalized with β-actin, respectively; and data are the mean ± SE. * p < 0.05 vs. the corresponding untreated control group. Un: Untreated cells.

Figure 5

Effects of ESWT on the expression of N-cadherin and E-cadherin in HTSFs. HTSFs were cultured from four patients with post burn hypertrophic scar tissue. The ESWT decreased the mRNA expression of N-cadherin (A), and increased the mRNA expression of E-cadherin (B). The mRNA expression was measured 24 h and 72 h after ESWT using a Light Cycler real-time PCR system. Each sample was assayed in duplicate, and experiments were performed least three times independently. The mRNA expression was normalized as ratio = 2^−∆∆Ct, and data are the mean ± S.E. * p < 0.05 and † p < 0.01 vs. the corresponding untreated control group. ESWT decreased the protein expression of N-cadherin (C), and increased protein expression of E-cadherin (D). Protein expression of N-cadherin and E-cadherin were measured with western blot analysis 24 h and 72 h after ESWT, respectively. The protein expression was normalized with β-actin, respectively, and data are the mean ± S.E. * p < 0.05 vs. the corresponding untreated control group. Un: Untreated cells.

Figure 6

ESWT regulates the expression of ID-1, ID-2, and twist-1 in HTSFs. HTSF was cultured from four patients with post burn hypertrophic scar tissue. The Protein expression of ID-1 (A), ID-2 (B) and (C) were measured with western blot analysis 24 h and 72 h after ESWT, respectively. The protein expression was normalized with lamin B1, and data are the mean ± S.E. * p < 0.05 vs. the corresponding untreated control group. Un: Untreated cells.

Figure 7

ESWT decreases migration of HTSFs. HTSFs were cultured from four patients with post burn hypertrophic scar tissue. (A) The HTSF cells seeded in a culture insert in a 35 mm µ-dish after ESWT, and then after 24 h, the culture insert was removed, made a cell-free gap, allow cells to migrate for 24 h and 48 h. The images were photographed under a light microscopy (scale bar, 500 µm). (B) Quantitative analysis of the migration assay was expressed as a percentage relative to untreated cells. The untreated cells were used as control, set to 100%. Data are the mean ± S.E. * p < 0.05 and † p < 0.01 vs. the corresponding untreated control group. Un: Untreated cells.