The Effects of Botulinum Toxin a on Collagen Synthesis, Expression Of

Archives of Aesthetic Plastic Surgery Vol. 19, No. 2, 114-119, 2013

The Effects of Botulinum Toxin A on Collagen Synthesis, Expression of MMP (matrix metalloproteinases)-1,2,9 and TIMP (tissue inhibitors of metalloproteinase)-1 in the Keloid Fibroblasts

Tai Suk Roh, M.D.1, Jong Won Hong, M.D.1, Won Jai Lee, M.D.2, Han-su Yoo, M.D.1, Dae Hyun Lew, M.D.2, and Young Seok Kim, M.D.1

1Department of Plastic & Reconstructive Surgery, Institute for Human Tissue Restoration, Gangnam Severance Hospital and 2Severance Hospital, Yonsei University College of Medicine, Seoul, Korea

Keloids are characterized by excessive extracelluar matrix (ECM) deposition such as collagen, fibronectin, Original Article elastin, and proteoglycans in the dermis. Recently, the use of botulinum toxin A (BTXA) in the treatment of keloids have had good results. To investigate the therapeutic effect of BTXA on the keloids, we evaluated the mRNA expression of collagen type I, type III, MMP (matrix metalloproteinases)-1, and TIMP (tissue inhibitor of metalloproteinases)-1 on keloid fibroblasts (KFs, n=5) after administration of BTXA. We also evaluated the enzymatic activity of MMP-2 and 9 by using zymography with BTXA. The same process was repeated after administration of TGF-ß in addition to BTXA. Type III collagen mRNA expression was decreased significantly when BTXA was administrated on KFs regardless of the presence or absence of TGF-ß. MMP-1 mRNA 114 expression in KFs was increased according to the BTXA concentration increment, however, not increased with TGF-ß. Moreover, MMP-2 enzymatic activity in KFs was increased when BTXA administrated regardless of the presence or absence of TGF-ß. These results suggest that the down regulation of collagen III expression, the up regulation of MMP-1, and increased MMP-2 enzymatic activity on KFs after BTXA administration are able to decrease the excess collagen deposition in keloids. (Arch Aesthetic Plast Surg 19: 114, 2013)

Key Words: Keloid, Botulinum toxins, Collagen type III, Matrix metalloproteinases, Tissue inhibitor of metalloproteinases Arch Aesthetic Plast Surg Arch I. INTRODUCTION pruritis, pain and hyperesthesia.2 They can cause functional im- pairment such as limited joint motion in addition to their poor Keloids are pathologic conditions resulting from excessive aesthetic appearance. Recently, the need of the keloid treatment extracelluar matrix (ECM) deposition in the dermis.1 They is increasing by these reasons. are often associated with clinical symptoms such as allodynia, Keloid fibroblasts (KFs) are not different from human normal dermal fibroblasts in size and shape, however, KFs can pro- Received June 8, 2013 liferate more than normal fibroblasts and produce high level of Revised June 27, 2013 ECM such as collagen, fibronectin, elastin, and proteoglycans.3-5 Accepted June 28, 2013 Keloids are also known as an abnormal balance between prolif- Address Correspondence: Young Seok Kim, M.D., Department 6 eration and apoptosis , which can lead to failure of homeostasis of Plastic & Reconstructive Surgery, Institute for Human Tissue Restoration, Gangnam Severance Hospital, Yonsei University, College in the wound healing process. of Medicine, 211 Eonjuro, Gangnam-gu, Seoul 135-720, Korea Elevated levels of Transforming growth factor (TGF)-β Tel: +82-2-2019-3420, Fax: 82-2-577-4914, E-mail:[email protected] and platelet derived growth factor (PDGF) have been found *The authors have no financial interests in any of the products, devices, or drugs mentioned in this article. in keloid tissue along with aberrant levels of their activity. *This work was supported by the Basic Science Research Program The elevated growth-factor activity is assumed that caused through the National Research Foundation of Korea (NRF), funded by increased expression of their respective receptors.7,8 TGF- by the Ministry of Education, Science, and Technology (2012- 0003180, YS Kim). β stimulates fibroblasts to produce and deposit collagen and Tai Suk Roh, et al.: The Effects of Botulinum Toxin A on Keloid Fibroblasts

ECM factors. TGF-β also induces production of PDGF, which Informed consent, which was approved by the Yonsei Uni- controls the rate of granulation tissue formation and stimulates versity College of Medicine Institutional Review Board, was collagen production during the later stages of wound healing.7 acquired from all patients. Keloids were diagnosed by plastic Furthermore, in the previous studies, the excessive ECM pro- surgeons and confirmed by pathologists. The Waymouth duction in keloids is related to an increase or decrease of matrix method was used for the tissue culture. The obtained tissue metalloproteinases (MMP-1, -2, -3, -9, -13) and an increase of was laid on petridish, and then washed twice with phosphate- tissue inhibitor of metalloproteinases (TIMPs).9-11 However, the buffered saline (PBS). A 7 ml of 0.25% trypsin solution was exact mechanism of keloids have not been known yet. equilibrated at 37℃. This solution added in the tissue, and then Symptomatic keloids have been treated in a variety ways, cells were isolated from it by Pasteur pipet. After 10 minutes, such as pressure dressings, antihistamine therapy, steroid the separated cells were cultured in 100 ml Dulbecco’s modi- injections,17 radiation therapy, chemotherapy (5-flurouracil, fied Eagle’s medium (DMEM; GIBCO, Grand Island, NY, imiquimod, interferon-alpha, verapamil etc)18 and surgical USA) supplemented with heat-inactivated 10% fetal bovine excision. Lasers, silicone, and topical retinoids also have been serum (FBS) and penicillin (30 U/ml), streptomycin (300 mg/ used.19 Besides the existing treatment options, botulinum toxin ml), and actinomysin, and then centrifuged by 3500RPM for 15 A (BTXA) has been used in the treatment of keloids, and a seconds. After the precipitates were removed, re-centrifugation good result of it have been reported.2,20,21 Clinical observations was done for 20 minutes to collect the next precipitates. The indicate that BTXA can improve the eventual appearance of collected precipitates were suspended in 5ml of the culture me- keloids, as inhibit the growth of keloids as well. However, the dium, and incubated in a humidified incubator at 37℃ and 5% mechanism of BTXA is unknown, aparted from the viewpoints CO2. The culture medium was changed in 2~3 day intervals. that BTXA improves keloids by decreasing tensile force of When primary cell culture reached confluent state, the culture muscle and skin near keloid tissue.22 medium was removed and washed with PBS. The cells on the MMP-2, -9 are known as gelatinases, which degrade dena- bottom of culture container were isolated completely with 2ml tured collagen (types I and III) fibrils, type IV collagen, fibronec- of 0.025% trypsin. Three to four times subcultred fibroblasts 115 tin and elastin. In addition, these enzymes can potentiate the were used in this study. The experimental group was added at degradation of ECM components by activating collagenase-3 various concentration of botulinum toxin A (BTXA) (Allergan (MMP-13) and neutrophil collagenase (MMP-8). Therefore, Corp., Irvine, CA, USA), and control group was not. gelatinases have an important role in physiologic soft tissue remodeling and pathologic wound healing.12,13 MMP-9 activity B. Reverse transcriptase-polymerase chain re- is crucial in the epithelization process, whereas MMP-2 activity action (RT-PCR) is important during the prolonged remodeling phase.14,15 The Total RNA of KFs was isolated by a Trizol reagent (Invitrogen MMP-2, -9 act on cleaved collagen more effectively than other Corp., Carlsbad, CA, USA) according to the manufacturer’s Arch Aesthetic Plast Surg Arch MMPs.12 MMP-1 is known for the capacity of reducing previ- protocol. One microgram of total RNA was converted into the ously formed ECM.16 For these reason, we hypothesized that complementary DNA using a First Strand cDNA Synthesis kit BTXA administration can affect molecular mechanisms of kel- (Promega Corp., Madison, WI, USA) with random primers. A oid such as collagen synthesis and enzymatic activity of MMP polymerase chain reaction was performed using 2 × Taq Premix or TIMP. In this study, we investigated the mRNA expression of 2 (Solgent Co., Ltd., Seoul, Korea) and the synthetic gene-spe- type I, III collagen synthesis, MMP-1 and TIMP-1 in KFs after cific primers for collagen types I and III, MMP-1, and TIMP-1. administration of various amounts of BTXA with or without Agarose gel electrophoresis was performed to separate samples TGF-β. We also evaluated enzymatic activity of MMP-2, 9 in of each reaction product, which was visualized by ethidium KFs with zymography after administration of various amounts bromide staining, and photographed with 290-nm ultraviolet il- of BTXA with or without TGF-β as well. lumination. The density of each band was measured by Image J.

II. MATERIALS AND METHODS C. MMP zymography Zymography was performed to evaluation of enzymatic A. Isolation and culture keloid-derived fibroblast activity of MMP-2 and MMP-9. Culture supernatants were culture collected and analyzed by gel substrate zymography. 10 mL Keloid-derived fibroblasts (KFs, n=5) were obtained from of non-reducing sample buffer (125 mM Tris-HCl [pH 6.8], the central dermal layer of keloid within 12 months of onset. 10% [v/v] glycerol, 0.1% [w/v] BPB) was added to each sample. Archives of Aesthetic Plastic Surgery Vol. 19, No. 2, 2013

12.5% SDS-PAGE gels containing 0.2% gelatin (Sigma, St. Lou- III. RESULTS is, MO, USA) was administrated in the samples. The gels were incubated with renaturing buffer (2.5% Triton X-100, 50mM A. The effect of BTXA on the mRNA expression Tris-HCl, pH 7.5, and 0.1M NaCl) for 1 hour at room tempera- of collagen type I and III in the KFs ture, in sequence with developing buffer (50 mM Tris-pH (pH The effect of BTXA on the collagen type I and III in the KFs

7.5), 10mM CaCl2, and 0.02% NaN3) for 18 hours at 37℃. The was analyzed by RT-PCR. Type I collagen mRNA expression gels were stained with 1% Coomassie brilliant blue R-250, and was not affected by BTXA administration, as well as by BTXA enzymatic activity of MMP-2, 9 were detected as unstained and TGF-β simultaneous administration. However, type III bands on a blue background. collage mRNA expression was decreased significantly (p<0.05) after administration of BTXA 0.5 unit/105 cells compared with 1) The effect of BTXA on the mRNA expression of control group. The type III collagen mRNA expression was de- collagen type I and III in the KFs creased significantly after TGF-β simultaneous administration Six plates were prepared from each samples (n=5), and each in addition to BTXA 0.5 unit/105 cells (Fig. 1). plate (total 30 plates) had 5x105 cultured KFs. In experimental group, KFs were cultured in DMEM with only Bovine Serum B. The effect of BTXA on the mRNA expression Albumin (BSA) for 24 hours before BTXA administration. of MMP-1 and TIMP-1 in the KFs The experimental group was treated with different amounts The effect of BTXA on MMP-1 and TIMP-1 mRNA expres- of BTXA (0.5 unit/105 cells, and 1.0 unit/105 cells), and the sion was analyzed by RT-PCR. BTXA increased mRNA ex- control group was not. The culture medium was collected at pression of MMP-1 significantly compared with control group 72 hours after the administration of BTXA. The Remnant cells (p<0.05). The expression degree of MMP-1 increased more as were washed with cold phosphate-buffered saline, and then BTXA concentration increased. (p<0.05) However, there was dissolved with lysis buffer solution. The cultured medium was not significantly increased expression of MMP-1 mRNA after 116 electrophoresed on sodium dodecyl sulfate-polyarylamide gels administration of TGF-β in addition to BTX. The mRNA ex- sequently. The RT-PCR was used for evaluation of mRNA expression of TIMP-1 was not affected by BTXA administration pression of collagen type I and III in the KFs. The same process regardless of TGF-β administration (Fig. 2). was repeated after administration of TGF-β (10 ng/mL). C. The effect of BTXA on MMP-2 and 9 enzy- 2) The effect of BTXA on the mRNA expression of matic activity in the KFs MMP-1 and TIMP-1 in the KFs The MMP-2 and 9 were known as gelatinase A and B respec- The same process mentioned previously was done. The tively, which break down ECM like other MMP as well. The mRNA expression of MMP-1 and TIMP-1 in KFs was analyzed enzymatic activity of MMP-2 and 9 were analyzed with zymog- Arch Aesthetic Plast Surg Arch by RT-PCR, and the same process was repeated after adminis- raphy. MMP-2 activity was significantly increased after BTXA tration of TGF-β (10 ng/mL). administration (p<0.05) whether TGF-β was administrated or not. (p<0.05) However, MMP-9 activity increased after BTXA 3) The effect of BTXA on MMP-2 and 9 enzymatic administration in comparison with the control group, but not activity in the KFs statistically significant (Fig. 3). The same process mentioned previously was done, and then enzymatic activity of MMP-2 and 9 with zymogram were IV. DISCUSSION analyzed quantitatively. The same process was repeated after administration of TGF-β (10 ng/mL) as well. Most therapeutic approaches for keloids remain clinically unsatisfactory, probably due to poor knowledge of the com- D. Statistics plex mechanisms underlying the process of excessive scarring. Statistical analysis between experimental and control group Hence, alternatives are needed.19,20,23,24 Recently, the use of were made using paired t-tests. Values were expressed as mean BTXA was suggested to extend the spectrum of treatment for ±standard deviation (SD), with statistical significance set at p keloids.2,25 Xiao et al., report that a flattening of the lesion and a <0.05. Data analysis relied on standard software PASW Statis- significant decrease in size was observed after BTXA injection tics 10.0 (SPSS, IBM, Inc, Chicago, IL, USA). in keloid.25 Uyesugi et al., declare that BTXA successfully treats the neuropathic symptoms associated with keloid scars.2 How- Tai Suk Roh, et al.: The Effects of Botulinum Toxin A on Keloid Fibroblasts

(A) (A)

(B) (B)

117

5 5 Aesthetic Plast Surg Arch 5 5 1: KFs(5x10 ) only 4: KFs(5x10 )+TGF-β 10ng/ml 1: KFs(5x10 ) only 4: KFs(5x10 )+TGF-β 10ng/ml 5 5 5 5 5 5 5 5 2: KFs(5x10 )+BTXA 0.5 U/10 cells 5: KFs(5x10 )+TGF-β 10ng/ml+BTXA 0.5U/10 cells 2: KFs(5x10 )+BTXA 0.5 U/10 cells 5: KFs(5x10 )+TGF-β 10ng/ml+BTXA 0.5U/10 cells 5 5 5 5 3: KFs(5x10 )+BTXA 1 U/10 cells 6: KFs(5x10 )+TGF-β 10ng/ml+BTXA 1U/10 cells 3: KFs(5x105)+BTXA 1 U/105 cells 6: KFs(5x105)+TGF-β 10ng/ml+BTXA 1U/105 cells Fig. 2. Fig. 1. The effect of Botulinum toxin A on the mRNA expression of The effect of Botulinum toxin A on the mRNA expression of MMP-1 and TIMP-1 in the keloid fibroblasts. (A): The effect of BTXA collagen type I and III in the keloid fibroblasts. (A): The effect of with or without TGF-β on the MMP-1 and TIMP-1 in the KFs was BTXA with or without TGF-β on the collagen type I and III in the analyzed by RT-PCR. Agarose gel electrophoresis was performed, and KFs was analyzed by RT-PCR. Agarose gel electrophoresis was per- the density of each band stood for mRNA expression of MMP-1 and formed, and the density of each band stood for mRNA expression of TIMP-1. (B): MMP-1 mRNA expression increased by BTXA signifi- collagen. (B): Type I collagen expression was not affected by BTXA cantly. However, this increase was disappeared after administration of and TGF-β. (C): Type III collagen mRNA expression significantly TGF-β in addition to BTXA. (C): TIMP-1 was not affected by admin- decrease in the keloid fibroblasts with BTXA 0.5 unit/105 cells. istration of BTXA with TGF-β or not (BTXA: botulinum toxin A). (p<0.05) After administration of BTXA 0.5 unit/105 cells and TGF-β simultaneously, type III collagen mRNA expression decreased significantly as well (col1: collagen type I, col3: collagen type III, BTXA: botulinum toxin A). nism of BTXA on keloid using cultured KFs. Keloids are characterized by an accumulation of collagen, it seems that the ever, most studies included a small number cases and lacked down regulation of collagen I and III expression and the up the experimental basis at the molecular or genetic level. They regulation of MMP are able to reduce the excess ECM deposi- cannot ascertain the detailed mechanism between BTXA and tion formed by the increase in synthetic activity. In this study, keloids as well. type III collagen mRNA expression was decreased significantly We investigated the potential underlying molecular mecha- when BTXA (0.5 unit/105 cells) was administrated on KFs Archives of Aesthetic Plastic Surgery Vol. 19, No. 2, 2013

study. Gauglitz et al, declared that BTXA injection did not result in keloid regression, and no differences in ECM marker expression, collagen synthesis, or TGF was observed after BTXA administration on KFs.20 However, they did not assess the change of MMP activity in KFs, and there are a lot of enzymes for ECM degradation and ECM markers involving keloid 5 5 1: KFs(5x10 ) only 4: KFs(5x10 )+TGF-β 10ng/ml growth and proliferation, so further experimental research was 2: KFs(5x105)+BTXA 0.5 U/105 cells 5: KFs(5x105)+TGF-β 10ng/ml+BTXA 0.5U/105 cells 3: KFs(5x105)+BTXA 1 U/105 cells 6: KFs(5x105)+TGF-β 10ng/ml+BTXA 1U/105 cells needed. A better understanding of the pathophysiology of keloid Fig. 3. Administration of BTXA to keloid fibroblasts, the enzymatic scarring hold great promise for developing novel therapeutic activity of MMP-2 and 9 were examined by zymography. MMP-2 activity increased significantly after administration of BTXA (p<0.05) strategies. In this regard, keloids are a result of excessive ac- and even after simultaneous administration of BTXA and TGF-β. cumulation of ECM which has be partly caused by increased (p<0.05) MMP-9 activity was not affected by BTXA and TGF-β. tensile force during the scar formation process. Additionally, the unbalance of cellular dynamics caused by the overabun- regardless of the presence or absence of TGF-β. Type I and III dance of cellular proliferation, and the lack of ECM degrada- collagens are accumulated high in keloid.24 It is the meaningful tion and cellular apoptosis plays a crucial role in the formation result that BTXA reduced type III collagen mRNA expression and growth of keloid. A new novel management for keloid with or without TGF-β in this study, even though did not effect treatment should target these findings. We can assume that on type I collagen mRNA expression. However, contrary to such a therapeutic effect (reduction of the tensile force during our hypothesis, type I and III collagen mRNA expression was around the keloid wound, as well as effective regulation of the more increased with administration of BTXA 1.0 unit/105 cells balance between cellular proliferation and apoptosis) could be than 0.5 unit/105 cells. For this reason, further evaluation for reached by BTXA.2,21,23 There are two possible hypotheses evi- 118 the therapeutic range of BTXA concentration has been needed. dences supporting the potential usefulness of BTXA for keloid MMP-1 mRNA expression in KFs was increased according to treatment. First, BTXA prevents contraction of muscle and the BTXA concentration increment, however, not increased skin near keloid tissue, which decrease tensile force during the with TGF-β in addition to BTXA. Moreover, MMP-2 enzy- course of cicatrization.2,21,23 Second, some research showed that matic activity in KFs was increased when BTXA administrated BTXA can influence on cellular dynamics and ECM degrada- regardless of the presence or absence of TGF-β. MMP-1 and tion.23 MMP-2 are the fibroblast collagenase and gelatinase A respec- We, therefore, can assume that this study presents experi- tively, and both break down ECM. mental data for the therapeutic effect of BTXA on keloid besides clinical studies. Arch Aesthetic Plast Surg Arch In addition, we expected that TIMP mRNA expression decreases after administration of BTXA, but no significant change was noted. We also expected that enzymatic activity of gelati- V. CONCLUSION nases (MMP-2, 9) increases with BTXA, but only MMP-2 activity increased according to the BTXA concentration. MMP-9 In this study, we could provide an experimental base of activity was not affected by BTXA and TGF-β. These may be BTXA for keloid management, and assume BTXA intralesional explained that BTXA selectively affect MMP-1 and 2 or BTXA injection will be a good alternative for keloid management as concentration of this study was not an effective range for TIMP well. and MMP-9. However, more work is needed to explain these results clearly. REFERENCES Moreover, some previous studies have shown that BTXA decreases mRNA expression of TGF-β in KFs.3,4 In this study, 1. Gauglitz GG, Korting HC, Pavicic T, Ruzicka T, Jeschke MG: Hypertro- after administration of BTXA with exogenous TGF-β, mRNA phic scarring and keloids: pathomechanisms and current and emerging treatment strategies. Mol Med 17: 113, 2011 expression of type III collage decreased and enzymatic activity 2. Uyesugi B, Lippincott B, Dave S: Treatment of a painful keloid with botu- of MMP-2 increased. These results may suggest that BTXA can linum toxin type A. 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