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Antimicrobial peptide KSL-W promotes gingival fibroblast healing properties in vitro
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Research Paper
Antimicrobial peptide KSL-W promotes gingival fibroblast healing properties in vitro Hyun-Jin Parka, Mabrouka Salema, Abdelhabib Semlalib, Kai P Leungc, Mahmoud Rouabhiaa,
⁎
a
Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec, QC, Canada Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia c Dental and Craniofacial Trauma Research and Tissue Regeneration Directorate, US Army Institute of Surgical Research, Joint Base Fort Sam Houston, TX 78234-6315, USA b
A R T I C L E I N F O
A B S T R A C T
Keywords: Antimicrobial peptide KSL-W Gingival fibroblasts F-actin Cell migration Cell cycle α-SMA
We investigated the effect of synthetic antimicrobial decapeptide KSL-W (KKVVFWVKFK) on normal human gingival fibroblast growth, migration, collagen gel contraction, and α-smooth muscle actin protein expression. Results show that in addition to promoting fibroblast adhesion by increasing F-actin production, peptide KSL-W promoted cell growth by increasing the S and G2/M cell cycle phases, and enhanced the secretion of metalloproteinase (MMP)-1 and MMP-2 by upregulating MMP inhibitors, such as tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in fibroblasts. An in vitro wound healing assay confirmed that peptide KSL-W promoted fibroblast migration and contraction of a collagen gel matrix. We also demonstrated a high expression of α-smooth muscle actin by gingival fibroblasts being exposed to KSL-W. This work shows that peptide KSL-W enhances gingival fibroblast growth, migration, and metalloproteinase secretion, and the expression of α-smooth muscle actin, thus promoting wound healing.
1. Introduction In recent years, antimicrobial peptides (AMPs) have generated much interest as an alternative anti-infective for the treatment of infections brought on by microbial resistance [1]. As part of the innate immune system of eukaryotes [2], AMPs generally display a broad spectrum of antimicrobial activity, with low cytotoxicity [1,2]. AMPs have been shown to be effective against bacterial pathogens as well as many different types of microbes, including protozoa, fungi, and even viruses [3]. For example, human defensins not only exhibit antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa [4], but also exert anti-human immunodeficiency virus-1 activity [5]. In general, resistance development is less likely to occur against AMPs because they kill bacteria by disrupting membrane integrity [6]. However, the challenges for AMPs have been their toxicity due to a relatively high dosages that are needed, the low selectivity for bacterial cell membrane, and their antimicrobial activity being reduced in the presence of salts under physiological conditions. There is also a concern about the short half-life in vivo due to rapid proteolytic cleavage. This may at least partly be connected to the large size of these natural AMPs [7,8]. To circumvent these limitations, short AMPs were designed and reported to be efficient against microorganisms, but with lower toxicity
against human cells [9–11]. Among these designed synthetic antimicrobial peptides, decapeptide KSL-W (KKVVFWVKFK) [12] displays improved stability in simulated oral conditions in vitro with preserved antimicrobial activity [12]. Moreover, KSL-W has been shown to inhibit the growth of various microorganisms [13,14]. In addition to their antibacterial activity, many AMPs reportedly possess immunomodulatory activity, such as an inhibition of lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production [15], and some of these peptides have been shown to stimulate angiogenesis, promote differentiation of immune cells, and possess wound healing properties [16]. Wound healing involves different cells, with epithelial cells and fibroblasts in the pole position of the wound healing process [17]. Indeed, epithelial cell migration is one of the major mechanisms for wound closing [15], and during wound healing; fibroblasts proliferate and migrate into the wound site to form new granulation tissue [18]. We know that cell migration is affected by cell-substrate interactions, specifically a stiffness of the surrounding and externally applied forces [19]. Fibroblasts produce new extracellular matrix (ECM) components for use as mechanically supportive structures. Proliferative fibroblasts known as myofibroblasts express high levels of α-smooth muscle actin (α-SMA) [20]. These cells generate remarkable contractile force which leads to wound contraction and remodeling as part of the healing
⁎ Corresponding author at: Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Pavillon de Médecine dentaire, 2420, rue de la Terrasse, Universit Laval Québec, Québec G1V 0A6, Canada. E-mail address: [email protected] (M. Rouabhia).
http://dx.doi.org/10.1016/j.peptides.2017.05.003 Received 8 February 2017; Received in revised form 1 May 2017; Accepted 6 May 2017 0196-9781/ © 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Park, H.-J., Peptides (2017), http://dx.doi.org/10.1016/j.peptides.2017.05.003
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field. Results are presented as the mean ± SD of four separate experiments.
process [21]. During ECM remodeling, balance is crucial between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) [22]. While TIMPs and MMPs are tightly regulated during normal wound healing, their imbalance has been reported following infection [23]. Because KSL-W has been shown to display antimicrobial and anti-fungal activity and because some AMPs show wound healing properties, we sought to determine whether antimicrobial peptide KSLW exerted any specific role in wound healing. Based on these premises, we investigated the effect of KSL-W on the wound healing process by assessing its contribution to human gingival fibroblast adhesion, growth, migration, and ability to contract collagen gel through the expression of α-SMA.
2.4. F-actin filament staining To confirm the attachment, we stained the F-actin filaments expressed by adherent fibroblasts. Following stimulation or not with peptide KSL-W, the cells were washed with PBS buffer, fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.5% (v/v) Triton X-100 in PBS for 10 min prior to staining. Non-specific binding was blocked by adding 1% (w/v) BSA for 30 min at room temperature. The slides were then washed and the F-actin fibers were stained using fluorescein isothiocyanate (FITC)-labelled phalloidin (Invitrogen, Molecular Probes). Phalloidin binds specifically to F-actin at the interface between F-actin subunits, locking adjacent subunits together, resulting in stabilizing actin filaments through the prevention of filament depolymerization [25]. Moreover, phalloidin reportedly inhibits the ATP hydrolysis activity of F-actin [26]. After three washes, the slides were visualized under a fluorescence microscope (Axiophot, Zeiss, Oberkochen, Germany) and digital images were collected (Coolpix 950, Nikon Canada, Montréal, QC, Canada).
2. Materials and methods 2.1. Culture of primary human gingival fibroblast cells Normal human gingival fibroblasts (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo Fisher Scientific, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Wisent Inc., Saint-Jean-Baptiste, QC, Canada). The medium was changed three times a week. When the culture reached 90% confluence, the cells were detached from the flasks with a 0.05% trypsin (MP Biomedicals LLC, Santa Ana, CA, USA)–0.1% ethylenediaminetetraacetic acid (EDTA) (Merck KGaA, Darmstadt, Germany) solution, washed twice with phosphate-buffered saline (PBS), and suspended thereafter in DMEM containing 10% FBS at a final concentration of 106 cells/mL. In general, cells at the third to the fifth passages were used to perform the experiments.
2.5. Effect of peptide KSL-W on the growth of primary human gingival fibroblasts Primary human gingival fibroblasts were seeded into 6-well plates (104 cells/well) in DMEM supplemented with 10% FBS and subsequently incubated in a 5% CO2 humid atmosphere at 37 °C for 24 h. Next, the cells were treated with 0, 10, 50, or 100 μg/mL of peptide KSL-W in fresh DMEM in duplicate for 3 and 6 days, respectively. The medium was changed every 24 h with fresh medium with or without KSL-W. At the end of the incubation, the cells were detached from the culture plates with a 0.05% trypsin/0.01% EDTA solution and were subsequently washed twice with culture medium, followed by a trypan blue exclusion assay to determine viable cell numbers. Briefly, 100 μL from each cell suspension were mixed with the same volume of the trypan blue (Sigma-Aldrich, Oakville, Ontario, Canada) solution (0.4%) and were incubated thereafter for 5 min on ice. Viable cells were determined in triplicate for each suspension by means of an inverted optical microscope to count the trypan blue-free cells. Results are reported as the mean ± SD of five separate experiments.
2.2. Antimicrobial peptide Peptide KSL-W was synthesized by standard solid-phase procedures with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry in an automatic peptide synthesizer (model 90, Advanced ChemTech, Louisville, KY, USA) [24]. The synthetic peptides were then purified by reverse-phase high-performance liquid chromatography (HPLC) (series 1100, Agilent Technologies, Santa Clara, CA, USA) employing a Vydac C18 column, with peptide purity confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) (AnaSpec, Fremont, CA, USA). The final product was stored in lyophilized format at −20 °C until use. A KSL-W solution was subsequently prepared, filtered (0.22 μm pore size), and used for the experiments.
2.6. Effect of KSL-W on the cell cycle status of primary human gingival fibroblasts
2.3. Effect of KSL-W on the attachment of primary human gingival fibroblasts
Primary human gingival fibroblasts were seeded into 6-well plates at 104 cells/well and then incubated for 48 h in a 5% CO2 humid atmosphere at 37 °C. Following incubation, the cells were placed in fresh medium with peptide KSL-W in duplicate at various concentrations of 0, 10, 50, or 100 μg/mL respectively. Post-treatment (24 h), the cells were detached from the culture plates as described above, washed twice with PBS, and subsequently subjected to cell cycle analysis. Briefly, the cells were suspended in an RNase (Promega, Madison, WI, USA) (10 mg/mL) solution and incubated thereafter at 37 °C for 1 h, followed by staining of the cells with propidium iodide (PI) (Abcam Inc., Toronto, ON, Canada) (50 mg/mL) prior to analysis. The percentage of cells in the G1, S, and G2/M phases of the cell cycle was determined by an Epics® Elite ESP flow cytometer (Beckman Coulter, Miami, FL, USA). The single cell population was gated using pulse width vs. pulse area to exclude clumps and doublets and the scatter plot was used to exclude any obvious debris. The PI was detected using a FL4 channel versus a cell count histogram plot. Results are reported as the mean ± SD of three separate experiments.
Prior to cell seeding, five sterile glass slides (Bellco Glass Inc., Vineland, NJ, USA) (0.05 mm in diameter) were inserted into each well of a non-adherent 6-well plate (Sarstedt, Nümbrecht, Germany). Primary human gingival fibroblasts were then seeded at 2 × 105 cells/well in DMEM supplemented with 10% FBS. Immediately after seeding, the cells were incubated with different concentrations (0, 10, 50, or 100 μg/mL) of peptide KSL-W in duplicate at 37 °C in a 5% CO2 incubator for 6 and 24 h, respectively. Following incubation, the cells were fixed with methanol (Fisher Scientific Co., Ottawa, ON, Canada) and glacial acetic acid (Merck KGaA) (75/25, v/v) for 15 min, followed by 3 washes with PBS. Thereafter, the fixed cells were incubated with 1 μg/mL Hoechst 33342 (H42) (Riedel de Haen, Seele, Germany) in PBS for 15 min at room temperature in the dark. After three washes with PBS, the samples were subjected to phase contrast microscopy and photographs were taken under an epifluorescence light microscope (Axiophot, Zeiss, Oberkochen, Germany). At least 10 fields from each slide were photographed and used to count the stained cells in each 2
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2.7. Cell migration/monolayer wound repair assay Primary human gingival fibroblasts were seeded (2 × 105/well) into 6-well plates and grown to confluence. A crossed scratch wound was created on each confluent monolayer by means of a 200 μL sterile pipette tip (PipetTipFinder, Lab Procurement Services, LLC, Knoxville, TN, USA) perpendicular to the bottom of the dish. This generated a wound approximately 0.44–0.50 mm in width [27]. The culture medium was then refreshed with new medium and the cells were incubated with or without peptide KSL-W at concentrations of 10, 50, or 100 μg/mL at 37 °C in a CO2 humid atmosphere. At the end of each incubation period (0, 12, 24, and 48 h), the control and peptideexposed cells were fixed with 4% paraformaldehyde solution (Alfa Aesar, Ward Hill, MA, USA) for 60 min at 4 °C and subjected thereafter to crystal violet staining (Sigma-Aldrich). Briefly, one milliliter of 1% w/v crystal violet solution in demineralized water was added to each well and the cultures were then incubated at room temperature for 15 min. The non-bound dye was removed from the wells by thorough washing with demineralized water, followed by drying at 37 °C. Digital photographs of each wound were taken (Coolpix 950, Nikon Canada) at various time points following the creation of the wound. Wound closure (cell migration) was investigated using the NIH Image J public domain image processing program to measure the distance between the opposite edges of the wound as a function of time. The distance between the wound edges was measured at least from 10 sites within the same wound. Data were presented as the percentage of initial wound (distance at time zero) using the following formula: [(distance at initial scratch − distance after individual treatment period) ÷ (distance at initial scratch)] × 100%. The KSL-W-treated and untreated cell cultures were compared, with the difference considered significant when p < 0.05. Results are reported as the mean ± SD of 5 separate experiments.
Fig. 1. Peptide KSL-W enhanced the early attachment of gingival fibroblasts. Cells were cultured with or without peptide KSL-W for 6 or 24 h and were then stained with Hoechst. The stained cells were counted and presented as the number of attached cells per microscope field. At least 10 fields from each slide were counted. P values were obtained by comparing the KSL-W-treated and untreated (Ctrl) values. Results are the mean + SD (n = 4).
Following collagen gel polymerization, 3 mL of culture medium with and without KSL-W (100 μg/mL) were added to each well, followed by incubation in a 5% CO2 humid atmosphere at 37 °C for various time periods. The contraction of each collagen gel specimen was measured by taking multiple measurements of the diameter. Results are reported as the mean ± SD of 4 separate experiments. 3. Effect of KSL-W on the expression of α-SMA by gingival fibroblasts Following culture in the presence or absence of KSL-W (100 μg/mL) for either 24 or 48 h, fibroblasts were detached from the culture plates. After 2 washes with PBS, cell lysates were prepared using a lysis buffer [25 mm of Tris–HCl, pH 8.0, 150 mm of NaCl, 1 mm of EDTA, 10% glycerol, 0.1% SDS, 0.05% sodium deoxycholate, 1% Triton X-100, and protease inhibitors (Sigma-Aldrich, cat. no. P2714) supplemented with anti-phosphatase cocktail III (Sigma-Aldrich, cat. no. P0044)]. The extracted protein concentrations were determined by the Bradford assay. Equal amounts of total protein (20–40 μg) in reducing sample buffer (61.5 mm of Tris, 100 mm of DTT, 2% SDS, and 10% glycerol) were boiled for 5 min and then electrophoresed by 10% SDS-PAGE. The proteins were transferred thereafter to PVDF membranes using a refrigerated Tris-glycine transfer buffer (25 mm of Tris, 192 mm of glycine, 100 μm of Na3VO4, and 20% methanol) for 1 h at 100 V. The blots were then incubated overnight at 4 °C with primary antibody against α-SMA (1∶100, clone 1A4, Sigma-Aldrich). β-actin (1:5000, A5441, Sigma-Aldrich), which exists in most cell type as component of the cytoskeleton, has been used as a housekeeping protein to correct for protein loading. Since both proteins (α-SMA and β-actin) have the same molecular weight (42 kDa), two membranes from the same samples with the same protein concentration were used to separately detect αSMA and β-actin. Finally, the membranes were washed and incubated for 1 h with the appropriate peroxidase-conjugated secondary antibodies. Detection was performed using the VersaDoc 5000MP Imaging System (Bio-Rad) and photographs were taken with Quantity One VersaDoc (Bio-Rad). Representative data out of three independent experiments were presented.
2.8. Effect of peptide KSL-W on the secretion of MMP-1, MMP-2, TIMP-1, and TIMP-2 by primary human gingival fibroblasts Supernatants were obtained from primary human gingival fibroblasts at 2 × 105/well in 6-well plates treated with 0, 10, 50, or 100 μg/mL of peptide KSL-W in duplicate for 3 and 6 days, respectively. MMP-1, MMP-2, TIMP-1, and TIMP-2 protein levels were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA, R & D Systems, Minneapolis, MN, USA) using the colleced supernatants. Briefly, the cell culture media were collected in tubes containing 1 μL of a protease inhibitor cocktail (Sigma-Aldrich) for specific use with mammalian cell and tissue extracts. The culture media were then filtered through 0.22μm filters and used to quantify the MMP-1, MMP-2, TIMP-1, and TIMP2 concentrations, according to the manufacturer’s instructions. Culture supernatants were diluted 1/100 (MMP-1 and TIMP-1), 1/50 (MMP-2) and 1/10 (TIMP-2) using sample dilluent solution as recommended by the manufacturer. The plates were read at 450 nm and analyzed thereafter by means of a Model 680 Microplate Reader (Bio-Rad, Hercules, CA, USA). According to the manufacturer, the minimum detectable concentrations were under 1 pg/mL for MMP-1, 0.7 pg/mL for MMP-2, 3.5 pg/mL for TIMP-1, and 3.5 pg/mL for TIMP-2. Results are reported as the mean ± SD of 4 separate experiments. 2.9. Effect of peptide KSL-W on collagen gel contraction by gingival fibroblasts The effect of KSL-W on the ability of human gingival fibroblasts to contract collagen gel was determined using a previously described reconstituted type I collagen assay system [28]. Both the untreated and KSL-W-treated fibroblasts were used to measure the collagen gel contraction. Briefly, fibroblasts (3 × 105) were mixed with rat tail collagen I (Cat. No. A 10483-01, Gibco, Invitrogen, Burlington, ON, Canada) and then poured onto 35 mm-diameter tissue culture plates.
3.1. Statistical analysis Each experiment was performed at least three independent times, with experimental values expressed as means ± SD. The statistical significance of the differences between the control (absence of KSL-W) and the test (presence of KSL-W) values was determined by means of a 3
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Fig. 2. F-actin expression following cell treatment or not with KSL-W. Cells were cultured with or without various concentrations of KSL-W for 6 and 24 h and were then stained with phalloidin-FITC. Note the well-organized, dense filamentous actin cytoskeleton (arrows) in the KSL-W-treated cells versus the untreated ones. Representative images are from 3 different experiments. Scale bars = 100 μm.
4. Results
one-way ANOVA. Posteriori comparisons were done using Tukey’s method. Normality and variance assumptions were verified using the Shapiro-Wilk test and the Brown and Forsythe test, respectively. All of the assumptions were fulfilled. P values were declared significant at ≤0.05. Data were analyzed using the SAS version 8.2 statistical package (SAS Institute Inc., Cary, NC, USA).
4.1. Peptide KSL-W promoted primary human gingival fibroblast attachment, F-actin filament expression, and cell growth Primary human gingival fibroblasts were treated with various concentrations of peptide KSL-W and subsequently analyzed to determine the effect of this antimicrobial peptide on their attachment and morphology. As shown in Fig. 1, our quantitative evaluation (numbering) of the attached cells revealed a higher cell number in the presence of peptide KSL-W than in the untreated control. We observe a 4
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over 7% of the cells were in the S phase, and close to 14% were in the G2/M phase (Fig. 4). However, following exposure to KSL-W, the G0/G1 phase values significantly (p < 0.05) decreased, and markedly so in the presence of 50 and 100 μg/mL of the peptide. In contrast, the S and G2/M phase values considerably (p < 0.05) increased in the presence of 100 μg/mL of KSL-W. Similar results were obtained with fibroblasts stimulated with KSL-W for 48 h (Fig. 4). 4.3. Peptide KSL-W increased MMP-1, MMP-2, TIMP-1, and TIMP-2 secretion by human gingival fibroblasts Results of the protein analyses of the spent culture media reveal that peptide KSL-W modulated the levels of remodeling enzyme proteins MMP-1 and MMP-2, as secreted by the gingival fibroblasts (Fig. 5). Indeed, increased levels of remodeling enzymes were observed after 3 and 6 days of peptide treatment, compared to that observed in the untreated cultures. Significant (p < 0.05) increases in MMP-1 levels were observed in the spent culture media from cells treated with 50 and 100 μg/mL of KSL-W for 3 days. Similar findings were recorded for the 6-day fibroblast culture treated with the same amounts of peptide. The levels of secreted MMP-1 were greater after 6 days than after 3 days, regardless of the test conditions (control or KSL-W-treated cultures). Comparable data were obtained for the MMP-2 protein (Fig. 5), as the recorded levels secreted from cells treated with 50 and 100 μg/mL of KSL-W for 3 and 6 days were significantly higher (p < 0.05). Because peptide KSL-W increased MMP-1 and MMP-2 secretion, we also investigated the peptide’s effect on TIMP-1 and TIMP2, which are involved in regulating MMP activities. Fig. 6A shows that peptide KSLW increased TIMP-1 secretion by the primary gingival fibroblasts. This increase was statistically significant (p < 0.05) in the presence of both 50 and 100 μg/mL of KSL-W and at both 3 and 6 days of stimulation with the peptide. We also analyzed the effect of KSL-W on TIMP-2 secretion (Fig. 6B). An increase in TIMP-2 secretion was recorded in the cells treated with all three tested concentrations of KSL-W only at day 6 (Fig. 6B).
Fig. 3. Peptide KSL-W increased gingival fibroblast growth. Fibroblasts were treated with or without various concentrations of peptide KSL-W for 3 and 6 days. Viable cells were analyzed by trypan blue exclusion assay. The numbers of live cells were plotted as the mean + SD (n = 5). P values were obtained by comparing the KSL-W-treated and untreated values. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
significant (p < 0.01) increase of cell adhesion with 50 and 100 μg/ mL of KSL-W at 6 h of culture. The attached cell per microscope field goes from 25 ± 2 cells with the control to 38 ± 8 with 50 μg/mL of KSL-W, and 45 ± 5 cells with 100 μg/mL of the peptide. Similar observation was found in the 24 h culture, showing a significant (p < 0.01) increase of attached cells. The cell number per microscope field increased from 50 ± 2 cells with the control to 70 ± 6 with 50 μg/mL of KSL-W, and 81 ± 9 cells with 100 μg/mL of the peptide (Fig. 1). Cell attachment was further confirmed by F-actin cytoskeleton staining. Fig. 2 shows attached cells expressing dense F-actin staining at as early as 6 h of culture. The staining intensity of the F-actin filaments was also stronger in the KSL-W-treated cells than in the untreated culture (Fig. 2, arrows). These observations were confirmed after 24 h of culture, with cells expressing a well-organized and dense filamentous F-actin cytoskeleton (Fig. 2, arrows) in the KSL-W-treated culture, compared to the untreated culture. As shown in Fig. 3, compared to the unstimulated primary human gingival fibroblast culture, our cell growth analysis revealed a trend showing a slight increase in the number of viable primary human gingival fibroblasts after 3 days, although this increase was not statistically significant. Compared to the control, however, the KSL-W (10 and 100 μg)-treated human fibroblasts recorded a much higher number of viable cells after 6 days, and this increased growth was statistically (p < 0.01) significant. At low concentration (10 μg/mL) the peptide KSL-W has no effect on gingival fibroblast growth. This increase in cell growth was also maintained in the presence of 50 or 100 μg/mL of KSL-W. Overall results show that peptide KSL-W promoted the growth of primary human gingival fibroblasts after 6 days.
4.4. Peptide KSL-W promoted cell migration and wound closure Because peptide KSL-W was found to promote fibroblast attachment and growth, we investigated its modulatory effect on fibroblast migration. Our results show that the fibroblasts treated with peptide KSL-W actively migrated from both edges and after 48 h had closed the entire wound (Fig. 7). This healing rate was much more rapid than that observed with the untreated cells which showed 25–30% less closure after 48 h (Fig. 7). Furthermore, a significant (p < 0.05) reduction of wound distance after 12 and 24 h was observed in the cells treated with 50 and 100 μg/mL of peptide KSL-W versus the untreated controls (Fig. 7). It appears that the increase in fibroblast migration was associated with higher peptide concentrations. 4.5. Peptide KSL-W promoted collagen gel contraction Collagen gel assay is one of several well-established in vitro assays to investigate cell behavior and three-dimensional (3D) matrix remodeling that closely resembles cell behavior in vivo. To establish whether KSL-W was beneficial to tissue repair by boosting the contractile capacity of fibroblasts, we investigated the collagen gel contraction in KSL-Wstimulated fibroblasts. As shown in Fig. 8, the gel contraction was greater in the presence of peptide KSL-W than in the control (absence of KSL-W stimulation). Our quantitative results show that from an initial dimeter of 5.5 cm, the collagen gel has contracted to 2.8 ± 0.05 cm diameter in the control and 2.3 ± 0.01 cm in the gel being treated with 100 μg/mL of KSL-W after 24 h (Fig. 8). After 48 h, the collagen gel diameter was 2.1 ± 0.17 cm in the control group and 1.6 ± 0.04 cm in the 100 μg/mL of KSL-W (Fig. 8). Overall data confirm that KSL-W augmented the contractile activity of the fibro-
4.2. Peptide KSL-W modulated the cell cycle progression of human gingival fibroblasts We speculated that the KSL-W-induced increase in cell growth possibly occurred through a modulation of the cell cycle progression. To monitor the effect of peptide KSL-W on the cell cycle, gingival fibroblasts were treated with various concentrations of KSL-W for 24 or 48 h and the number of cells were quantified at different cell cycle phases (Fig. 4). Thus in the KSL-W-untreated group after 24 h of culture, approximately 70% of the cells were in the G0/G1 phase, while 5
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Fig. 4. Peptide KSL-W promoted cell division. Following stimulation for 24 and 48 h, the cells were detached and used for cell cycle testing by PI staining. Quantitation of the cell percentages at the different phases was determined by FAC analysis. P < 0.05 was obtained by comparing the KSL-W-treated and untreated cells (n = 3).
blasts, which may be explained by the expression of contractile fibers, such as α-SMA fibers. Compared to the untreated control cells, as demonstrated by Western blot analyses, the KSL-W-treated fibroblasts showed an increased expression of α-SMA contractile fibers (a stronger intensity of the protein band corresponding to α-SMA) following both 24 and 48 h of exposure to KSL-W (100 μg/mL) (Fig. 9).
cationic surface-active agent [31], peptide KSL-W has been shown to significantly reduce oral biofilm growth in vitro [32]. In addition to the inherent ability of antimicrobial peptides to kill bacteria, some of these peptides, show wound healing properties [33]. LL37, for example, at low concentration (5 μg/mL) stimulated airway epithelial cell proliferation and wound closure, while it induced necrosis at high concentration (> 20 μg/mL) [34]. We therefore sought to study the effect of peptide KSL-W on primary human gingival fibroblasts which are among the important wound healing cell types. Our results indicate that this antimicrobial peptide was non-toxic, even at a higher concentration (100 μg/mL), not like LL-37 [34], and was able to stimulate fibroblast growth. Nisin Z, an antimicrobial peptide [35] has been shown to enhance fibroblast defenses against infection [28]. Similarly, other antimicrobial peptides, in addition to their inherent antimicrobial activity, have been shown to stimulate the growth and migration of HaCat cells and human dermal fibroblasts
5. Discussion Various antimicrobial peptides are known to display in vitro activity against a wide range of bacterial pathogens and are thus being proposed as alternatives to develop novel antibacterial disease-control strategies [3,29]. Among available antimicrobial peptides, peptide KSL-W has demonstrated improved stability in simulated oral conditions with broad spectrum antimicrobial activity [30]. Furthermore, combined with sub-inhibitory concentrations of benzalkonium chloride, a known 6
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Fig. 6. Peptide KSL-W enhanced metalloproteinase inhibitor (TIMP-1 and TIMP-2) secretion. Note the increased levels of TIMPs with the high doses of peptide KSL-W after 6 days of stimulation. P < 0.05 was obtained by comparing the KSL-W-treated and untreated cells and the levels after 3 and 6 days (n = 4).
Fig. 5. Peptide KSL-W increased metalloproteinase (MMP-1 and MMP-2) secretion. Fibroblasts were stimulated for 3 and 6 days with different concentrations of KSL-W. Cell supernatants were used to measure the MMPs by specific ELISA. Note the increased MMP levels with the high doses of peptide KSL-W. P < 0.05 was obtained by comparing the KSL-W-treated and untreated (Ctrl) cells and the levels after 3 and 6 days (n = 4).
levels by human gingival fibroblasts. Similar observations were reported with α-defensin-1, shown to cause increased levels of mRNA expression of IL-6, IL-8, MMP-1, and MMP-3 by fibroblast-like synoviocytes [40]. In our study, when gingival fibroblasts were exposed to peptide KSL-W, the increased MMP secretion was paralleled by TMIP-1 and TIMP-2 secretion. Overall, peptide KSL-W stimulation enhanced fibroblast growth and increased MMP and TIMP secretion, which suggests that this peptide may contribute to cell migration and wound healing. To test this hypothesis, KSL-W-stimulated gingival fibroblasts were subjected to an in vitro wound scratch assay which showed that peptide KSL-W at 50 and 100 μg/mL promoted fibroblast migration and wound closure. These findings are in agreement with those of earlier studies showing that naturel antimicrobial peptides, such as human β-defensins-2, promoted cell migration and proliferation [41] and significantly accelerated wound closure when topically applied in a porcine model of infected skin wounds [42]. Studies have suggested that antimicrobial peptides on wounds serve as a modulator to help cells migrate during the wound healing process [43]. Essential molecules, such as epidermal growth factor receptor, STAT3 protein, and mitogen-activated protein (MAP)-kinases, have been shown to be involved in antimicrobial peptide-induced cellular reactions [43]. Because of the participation of these signaling molecules associated with cell activation and differentiation, we hypothesized that KSL-Wtreated fibroblasts may be involved in wound closure through their activation/differentiation into myofibroblasts. These cells are key players in the wound healing process and are responsible for cell-
[36]. Another study evidenced that ocellatin peptides extracted from frog Leptodactylus pustulatus skin controlled microbial growth with no adverse effect on human erythrocytes and a murine fibroblast cell line [37]. In our study, the effect of peptide KSL-W on fibroblast growth appears to have occurred through its influence on the cell cycle, as the KSL-W-treated cell cultures recorded a greater number of cells at the S and G2/M phases. As for the molecular target(s) of peptide KSL-W contributing to increased fibroblast growth in the treated gingival fibroblasts, this remains unclear. This effect is comparable to that of naturally occurring peptide catestatin secreted by human keratinocytes. Hoq et al. [38] showed that in addition to inhibiting the growth of pathogens, catestatin peptides promoted keratinocyte proliferation. Because fibroblasts adhered and proliferated in the presence of KSLW, this may thus have increased their physiological activity by stimulating their secretion of both MMP secretion inhibitors (TIMPs), as observed in our study. Similar to what has been reported with β-defensin-3 [39], the increased secretion of MMP-1 and MMP-2 by the KSL-W-treated fibroblasts may point to the involvement of peptide KSL-W in mediating part of the tissue remodeling processes. Alternatively, changes in MMPs could be considered as an inflammatory response by fibroblasts against the presence of peptide KSL-W, as was demonstrated [39] with other antimicrobial peptides, such as human β-defensin-3, which was shown to cause an increase in prostaglandin-(PGE) 2 and MMP-1 secretion 7
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Fig. 7. Peptide KSL-W increased gingival fibroblast migration/repair. Cells were cultured up to 100% confluence. Scratches were then made on each monolayer and the medium was refreshed and immediately treated with or without peptide KSL-W at different concentrations. The cultures were maintained for various time periods prior to observation and determination of the wound recovery. (A) Representative Photos 12 and 48 h post-wound (bar 50 μm). (B) Values are expressed as the mean ± SD (n = 5). The KSL-W-treated and untreated cultures were compared, with the difference considered significant at (*) p < 0.05.
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Fig. 8. Gingival fibroblasts stimulated with KSL-W contracted the collagen gel matrix. Following inclusion into the collagen matrix, fibroblasts were cultured in the presence or absence of KSL-W (100 μg/mL). The diameter of each collagen gel was measured after 24 and 48 h. Representative photos show the action of the KSL-W-treated and untreated cells on collagen gel contraction. The diameters of the collagen gels are presented. Values are expressed as the mean ± SD (n = 5). The KSL-W-treated and untreated tissues were compared, with the difference considered significant at p < 0.05.
These findings concur with others showing the ability of antimicrobial peptide LLKKK18 to accelerate wound closure and simultaneously improve the quality of healing [45]. Wound contraction occurred by the expression of α-SMA, which is critical to wound contraction and the differentiation marker of myofiboblasts from fibroblasts [46–48]. Concordantly, the KSL-W-enhanced fibroblast migration and collagen gel contraction shown in our study can be explained by the significant expression of α-SMA by the fibroblasts following stimulation with the peptide (100 μg/mL). Our study suggests a role of antimicrobial peptide in wound healing mechanisms through α-SMA. Our data suggest that one possible way by which antimicrobial peptide KSL-W helps wound healing is by increasing α-SMA expression in fibroblasts. This finding is significant for clinical application, with antimicrobial peptides being used to not only control infection but also contribute to the wound healing process.
Fig. 9. KSL-W increased α-SMA protein expression by gingival fibroblasts. Following cell stimulation with KSL-W (100 μg/mL) or not, proteins were extracted and analyzed by Western blot. Results show greater α-SMA expression in the KSL-W-treated cells than in the untreated cells.
mediated matrix contraction [44]. Through the contraction of their actin cytoskeleton, myofibroblasts at the wound site are able to reduce the initial size of the wound, thereby contributing to tissue repair [44]. In light of this, our study demonstrated that KSL-W-stimulated fibroblasts were able to induce the contraction of collagen gel matrix.
6. Conclusion Our study shows that antimicrobial peptide KSL-W promoted 9
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human fibroblast growth by increasing the cell population in the S and G2/M phases of the cell cycle. KSL-W also promoted the secretion of matrix metalloproteinase and tissue inhibitors of metalloproteinase, TIMP1 and TIMP2. Furthermore, this antimicrobial peptide increased fibroblast migration and collagen gel contraction in vitro, which may be due to the α-SMA protein expression and contraction as the results of the fibroblast-myofibroblast transdifferentiation induced by the peptide. Increase in α-SMA protein expression was observed in the KSL-Wtreated fibroblasts. Overall data suggest that KSL-W may have wound healing properties.
(2007) 1544–1550. [13] D.R. Dixon, L. Karimi-Naser, R.P. Darveau, K.P. Leung, The anti-endotoxic effects of the KSL-W decapeptide on Escherichia coli O55:B5 and various oral lipopolysaccharides, J. Periodontal Res. 43 (2008) 422–430. [14] A. Semlali, K.P. Leung, S. Curt, M. Rouabhia, Antimicrobial decapeptide KSL-W attenuates Candida albicans virulence by modulating its effects on Toll-like receptor, human β-defensin, and cytokine expression by engineered human oral mucosa, Peptides 32 (2011) 859–867. [15] A. Pfalzgraff, L. Heinbockel, Q. Su, T. Gutsmann, K. Brandenburg, G. Weindl, Synthetic antimicrobial and LPS-neutralising peptides suppress inflammatory and immune responses in skin cells and promote keratinocyte migration, Sci. Rep. 6 (2016) 31577. [16] R.E. Hancock, E.F. Haney, E.E. Gill, The immunology of host defence peptides: beyond antimicrobialactivity, Nat. Rev. Immunol. 16 (2016) 321–334. [17] L.E. Tracy, R.A. Minasian, E.J. Caterson, Extracellular Matrix and Dermal Fibroblast Function in the HealingWound, Adv. Wound Care (New Rochelle) 5 (2016) 119–136. [18] S. Toume, A. Gefen, D. Weihs, Low-level stretching accelerates cell migration into a gap, Int. Wound J. (October (17)) (2016), http://dx.doi.org/10.1111/iwj.12679. [19] O. Yamanaka, S. Saika, K. Ikeda, K. Miyazaki, A. Kitano, Y. Ohnishi, Connective tissue growth factor modulates extracellular matrix production in human subconjunctival fibroblasts and their proliferation and migration in vitro, Jpn. J. Ophthalmol. 52 (2008) 8–15. [20] Y.B. Kwon, H.W. Kim, D.H. Roh, S.Y. Yoon, R.M. Baek, J.Y. Kim, et al., Topical application of epidermal growth factor accelerates wound healing by myofibroblast proliferation and collagen synthesis in rat, J. Vet. Sci. 7 (2006) 105–109. [21] I.A. Darby, N. Zakuan, F. Billet, A. Desmoulière, The myofibroblast, a key cell in normal and pathological tissue repair, Cell. Mol. Life Sci. 73 (2016) 1145–1157. [22] A. 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Garrido, et al., Peptide KSL-W-Loaded mucoadhesive liquid crystalline vehicle as an alternative treatment for multispecies oral biofilm, Molecules 21 (2015) 14. [33] A. Gronberg, M. Mahlapuu, M. Stahle, C. Whately-Smith, O. Rollma, Treatment with LL-37 is safe and effective in enhancing healing of hard-to-heal venous leg ulcers: a randomized, placebo-controlled clinical trial, Wound Repair Regen. 22 (2014) 613–621. [34] R. Shaykhiev, C. Beisswenger, K. Kändler, J. Senske, A. Püchner, T. Damm, J. Behr, R. Bals, Human endogenous antibiotic LL-37 stimulates airway epithelial cell proliferation and wound closure, Am. J. Physiol. Lung Cell. Mol. Physiol. 289 (5) (2005) L842–8. [35] B. Akerey, C. Le-Lay, I. Fliss, M. Subirade, M. Rouabhia, In vitro efficacy of nisin Z against Candida albicans adhesion and transition following contact with normal human gingival cells, J. Appl. Microbiol. 107 (2009) 1298–1307. [36] P. Kosikowska, M. Pikula, P. Langa, P. Trzonkowski, M. Obuchowski, A. Lesner, Synthesis and evaluation of biological activity of antimicrobial–pro-proliferative peptide conjugates, PLoS One 10 (2015) e0140377. [37] M.M. Marani, F.S. Dourado, P.V. Quelemes, A.R. de Araujo, M.L. Perfeito, E.A. Barbosa, et al., Characterization and biological activities of ocellatin peptides from the skin secretion of the frog leptodactylus pustulatus, J. Nat. Prod. 78 (2015) 1495–1504. [38] M.I. Hoq, F. Niyonsaba, H. Ushio, G. Aung, K. Okumura, H. Ogawa, Human catestatin enhances migration and proliferation of normal human epidermal keratinocytes, J. Dermatol. Sci. 64 (2011) 108–118. [39] C.X. Jin, Z.X. Qiao, B. Liu, D.L. Chen, Y.L. Wang, Human β-defensin-3 regulates the proliferation and the secretion of prostaglandin E2 and matrix metalloproteinase-1 in human gingival fibroblasts, Zhonghua Kou Qiang Yi Xue Za Zhi 48 (2013) 734–739. [40] J.K. Ahn, B. Huang, E.K. Bae, E.J. Park, J.W. Hwang, J. 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Authors’ contributions MR, KPL, and AS designed the experiments, supervised the research, and wrote the paper. HJP, MS, AS, and MR performed the experiments and data analyses. Each author read and approved the submission of the final version of the manuscript. DOD disclaimer One of the authors (KPL) is a United States Government employee. The work presented is part of his official duties. The opinions or assertions contained herein are the personal views of these authors and are not to be construed as official or reflecting the views of the United States Army or Department of Defense. Ethical approval Not required. Acknowledgements: This study was supported by funding from the United States Army Medical Research and Materiel Command (Award number ERMS No. 12304006) and by a grant from the Fonds Émile-Beaulieu, at Université Laval foundation. The authors wish to thank Dr. Haldar Subrata for the review and the discussion of the manuscript. References [1] S. Ramesh, T. Govender, H.G. Kruger, B.G. de la Torre, F. Albericio, Short AntiMicrobial Peptides (SAMPs) as a class of extraordinary promising therapeutic agents, J. Pept. Sci 22 (2016) 438–451. [2] M. Pasupuleti, A. Schmidtchen, M. Malmsten, Antimicrobial peptides: key components of the innate immune system, Crit. Rev. Biotechnol. 32 (2012) 143–171. [3] F. Findlay, L. Proudfoot, C. Stevens, P.G. Barlow, Cationic host defense peptides; novel antimicrobial therapeutics against Category A pathogens and emerging infections, Pathog. Glob. Health 110 (2016) 137–147. [4] L.C. Huang, R.L. Redfern, S. Narayana, R.Y. Reins, A.M. McDermott, In vitro activity of human beta-defensin 2 against Pseudomonas aeruginosa in the presence of tear fluid, Antimicrob. Agents Chemother. 51 (2007) 3853–3880. [5] L. Furci, M. Tolazzi, F. Sironi, L. Vassena, P. Lusso, Inhibition of HIV-1 infection by human α-defensin-5, a natural antimicrobial peptide expressed in the genital and intestinal mucosae, PLoS One 7 (2012) e45208. [6] K.A. Brogden, Antimicrobial peptides: pore formers or metabolic inhibitors in bacteria? Nat. Rev. Microbiol. 3 (2005) 238–250. [7] C.D. Fjell, J.A. Hiss, R.E. Hancock, G. Schneider, Designing antimicrobial peptides: form follows function, Nat. Rev. Drug Discov. 11 (1) (2012) 37–51. [8] M. Zaiou, Multifunctional antimicrobial peptides: therapeutic targets in several human diseases? J Mol Med (Berl) 85 (4) (2007) 317–329. [9] B.E. Haug, W. Stensen, M. Kalaaji, Ø. Rekdal, J.S. Svendsen, Synthetic antimicrobial peptidomimetics with therapeutic potential, J. Med. Chem. 51 (14) (2008) 4306–4314. [10] P.F. Kreling, K.L. Aida, L. Massunari, K.S. Caiaffa, C. Percinoto, T.B.L. Bedran, D.M.P. Spolidorio, G.F. Abuna, E.M. Cilli, C. Duque, Cytotoxicity and the effect of cationic peptide fragments against cariogenic bacteria under planktonic and biofilm conditions, Biofouling 32 (9) (2016) 995–1006. [11] M. Taniguchi, A. Ochiai, K. Takahashi, S.-I. Nakamichi, T. Nomoto, E. Saitoh, T. Kato, T. Tanaka, Antimicrobial activity and mechanism of action of a novel cationic α-helical octadecapeptide derived from α-amylase of rice, Biopolymers 104 (2) (2015) 73–83. [12] D.H. Na, J. Faraj, Y. Capan, K.P. Leung, P.P. DeLuca, Stability of antimicrobial decapeptide (KSL) and its analogues for delivery in the oral cavity, Pharm. Res. 24
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Peptides journal homepage: www.elsevier.com/locate/peptides
Research Paper
Antimicrobial peptide KSL-W promotes gingival fibroblast healing properties in vitro Hyun-Jin Parka, Mabrouka Salema, Abdelhabib Semlalib, Kai P Leungc, Mahmoud Rouabhiaa,
⁎
a
Groupe de Recherche en Écologie Buccale, Faculté de Médecine Dentaire, Université Laval, Québec, QC, Canada Department of Biochemistry, College of Science, King Saud University, Riyadh, Saudi Arabia c Dental and Craniofacial Trauma Research and Tissue Regeneration Directorate, US Army Institute of Surgical Research, Joint Base Fort Sam Houston, TX 78234-6315, USA b
A R T I C L E I N F O
A B S T R A C T
Keywords: Antimicrobial peptide KSL-W Gingival fibroblasts F-actin Cell migration Cell cycle α-SMA
We investigated the effect of synthetic antimicrobial decapeptide KSL-W (KKVVFWVKFK) on normal human gingival fibroblast growth, migration, collagen gel contraction, and α-smooth muscle actin protein expression. Results show that in addition to promoting fibroblast adhesion by increasing F-actin production, peptide KSL-W promoted cell growth by increasing the S and G2/M cell cycle phases, and enhanced the secretion of metalloproteinase (MMP)-1 and MMP-2 by upregulating MMP inhibitors, such as tissue inhibitors of metalloproteinase (TIMP)-1 and TIMP-2 in fibroblasts. An in vitro wound healing assay confirmed that peptide KSL-W promoted fibroblast migration and contraction of a collagen gel matrix. We also demonstrated a high expression of α-smooth muscle actin by gingival fibroblasts being exposed to KSL-W. This work shows that peptide KSL-W enhances gingival fibroblast growth, migration, and metalloproteinase secretion, and the expression of α-smooth muscle actin, thus promoting wound healing.
1. Introduction In recent years, antimicrobial peptides (AMPs) have generated much interest as an alternative anti-infective for the treatment of infections brought on by microbial resistance [1]. As part of the innate immune system of eukaryotes [2], AMPs generally display a broad spectrum of antimicrobial activity, with low cytotoxicity [1,2]. AMPs have been shown to be effective against bacterial pathogens as well as many different types of microbes, including protozoa, fungi, and even viruses [3]. For example, human defensins not only exhibit antimicrobial activity against Staphylococcus aureus and Pseudomonas aeruginosa [4], but also exert anti-human immunodeficiency virus-1 activity [5]. In general, resistance development is less likely to occur against AMPs because they kill bacteria by disrupting membrane integrity [6]. However, the challenges for AMPs have been their toxicity due to a relatively high dosages that are needed, the low selectivity for bacterial cell membrane, and their antimicrobial activity being reduced in the presence of salts under physiological conditions. There is also a concern about the short half-life in vivo due to rapid proteolytic cleavage. This may at least partly be connected to the large size of these natural AMPs [7,8]. To circumvent these limitations, short AMPs were designed and reported to be efficient against microorganisms, but with lower toxicity
against human cells [9–11]. Among these designed synthetic antimicrobial peptides, decapeptide KSL-W (KKVVFWVKFK) [12] displays improved stability in simulated oral conditions in vitro with preserved antimicrobial activity [12]. Moreover, KSL-W has been shown to inhibit the growth of various microorganisms [13,14]. In addition to their antibacterial activity, many AMPs reportedly possess immunomodulatory activity, such as an inhibition of lipopolysaccharide (LPS)-induced pro-inflammatory cytokine production [15], and some of these peptides have been shown to stimulate angiogenesis, promote differentiation of immune cells, and possess wound healing properties [16]. Wound healing involves different cells, with epithelial cells and fibroblasts in the pole position of the wound healing process [17]. Indeed, epithelial cell migration is one of the major mechanisms for wound closing [15], and during wound healing; fibroblasts proliferate and migrate into the wound site to form new granulation tissue [18]. We know that cell migration is affected by cell-substrate interactions, specifically a stiffness of the surrounding and externally applied forces [19]. Fibroblasts produce new extracellular matrix (ECM) components for use as mechanically supportive structures. Proliferative fibroblasts known as myofibroblasts express high levels of α-smooth muscle actin (α-SMA) [20]. These cells generate remarkable contractile force which leads to wound contraction and remodeling as part of the healing
⁎ Corresponding author at: Groupe de recherche en écologie buccale, Faculté de médecine dentaire, Pavillon de Médecine dentaire, 2420, rue de la Terrasse, Universit Laval Québec, Québec G1V 0A6, Canada. E-mail address: [email protected] (M. Rouabhia).
http://dx.doi.org/10.1016/j.peptides.2017.05.003 Received 8 February 2017; Received in revised form 1 May 2017; Accepted 6 May 2017 0196-9781/ © 2017 Elsevier Inc. All rights reserved.
Please cite this article as: Park, H.-J., Peptides (2017), http://dx.doi.org/10.1016/j.peptides.2017.05.003
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field. Results are presented as the mean ± SD of four separate experiments.
process [21]. During ECM remodeling, balance is crucial between matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) [22]. While TIMPs and MMPs are tightly regulated during normal wound healing, their imbalance has been reported following infection [23]. Because KSL-W has been shown to display antimicrobial and anti-fungal activity and because some AMPs show wound healing properties, we sought to determine whether antimicrobial peptide KSLW exerted any specific role in wound healing. Based on these premises, we investigated the effect of KSL-W on the wound healing process by assessing its contribution to human gingival fibroblast adhesion, growth, migration, and ability to contract collagen gel through the expression of α-SMA.
2.4. F-actin filament staining To confirm the attachment, we stained the F-actin filaments expressed by adherent fibroblasts. Following stimulation or not with peptide KSL-W, the cells were washed with PBS buffer, fixed with 4% paraformaldehyde for 15 min, and then permeabilized with 0.5% (v/v) Triton X-100 in PBS for 10 min prior to staining. Non-specific binding was blocked by adding 1% (w/v) BSA for 30 min at room temperature. The slides were then washed and the F-actin fibers were stained using fluorescein isothiocyanate (FITC)-labelled phalloidin (Invitrogen, Molecular Probes). Phalloidin binds specifically to F-actin at the interface between F-actin subunits, locking adjacent subunits together, resulting in stabilizing actin filaments through the prevention of filament depolymerization [25]. Moreover, phalloidin reportedly inhibits the ATP hydrolysis activity of F-actin [26]. After three washes, the slides were visualized under a fluorescence microscope (Axiophot, Zeiss, Oberkochen, Germany) and digital images were collected (Coolpix 950, Nikon Canada, Montréal, QC, Canada).
2. Materials and methods 2.1. Culture of primary human gingival fibroblast cells Normal human gingival fibroblasts (ScienCell Research Laboratories, Carlsbad, CA, USA) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Gibco, Thermo Fisher Scientific, Gaithersburg, MD, USA) supplemented with 10% fetal bovine serum (FBS) (Wisent Inc., Saint-Jean-Baptiste, QC, Canada). The medium was changed three times a week. When the culture reached 90% confluence, the cells were detached from the flasks with a 0.05% trypsin (MP Biomedicals LLC, Santa Ana, CA, USA)–0.1% ethylenediaminetetraacetic acid (EDTA) (Merck KGaA, Darmstadt, Germany) solution, washed twice with phosphate-buffered saline (PBS), and suspended thereafter in DMEM containing 10% FBS at a final concentration of 106 cells/mL. In general, cells at the third to the fifth passages were used to perform the experiments.
2.5. Effect of peptide KSL-W on the growth of primary human gingival fibroblasts Primary human gingival fibroblasts were seeded into 6-well plates (104 cells/well) in DMEM supplemented with 10% FBS and subsequently incubated in a 5% CO2 humid atmosphere at 37 °C for 24 h. Next, the cells were treated with 0, 10, 50, or 100 μg/mL of peptide KSL-W in fresh DMEM in duplicate for 3 and 6 days, respectively. The medium was changed every 24 h with fresh medium with or without KSL-W. At the end of the incubation, the cells were detached from the culture plates with a 0.05% trypsin/0.01% EDTA solution and were subsequently washed twice with culture medium, followed by a trypan blue exclusion assay to determine viable cell numbers. Briefly, 100 μL from each cell suspension were mixed with the same volume of the trypan blue (Sigma-Aldrich, Oakville, Ontario, Canada) solution (0.4%) and were incubated thereafter for 5 min on ice. Viable cells were determined in triplicate for each suspension by means of an inverted optical microscope to count the trypan blue-free cells. Results are reported as the mean ± SD of five separate experiments.
2.2. Antimicrobial peptide Peptide KSL-W was synthesized by standard solid-phase procedures with 9-fluorenylmethoxycarbonyl (Fmoc) chemistry in an automatic peptide synthesizer (model 90, Advanced ChemTech, Louisville, KY, USA) [24]. The synthetic peptides were then purified by reverse-phase high-performance liquid chromatography (HPLC) (series 1100, Agilent Technologies, Santa Clara, CA, USA) employing a Vydac C18 column, with peptide purity confirmed by MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) (AnaSpec, Fremont, CA, USA). The final product was stored in lyophilized format at −20 °C until use. A KSL-W solution was subsequently prepared, filtered (0.22 μm pore size), and used for the experiments.
2.6. Effect of KSL-W on the cell cycle status of primary human gingival fibroblasts
2.3. Effect of KSL-W on the attachment of primary human gingival fibroblasts
Primary human gingival fibroblasts were seeded into 6-well plates at 104 cells/well and then incubated for 48 h in a 5% CO2 humid atmosphere at 37 °C. Following incubation, the cells were placed in fresh medium with peptide KSL-W in duplicate at various concentrations of 0, 10, 50, or 100 μg/mL respectively. Post-treatment (24 h), the cells were detached from the culture plates as described above, washed twice with PBS, and subsequently subjected to cell cycle analysis. Briefly, the cells were suspended in an RNase (Promega, Madison, WI, USA) (10 mg/mL) solution and incubated thereafter at 37 °C for 1 h, followed by staining of the cells with propidium iodide (PI) (Abcam Inc., Toronto, ON, Canada) (50 mg/mL) prior to analysis. The percentage of cells in the G1, S, and G2/M phases of the cell cycle was determined by an Epics® Elite ESP flow cytometer (Beckman Coulter, Miami, FL, USA). The single cell population was gated using pulse width vs. pulse area to exclude clumps and doublets and the scatter plot was used to exclude any obvious debris. The PI was detected using a FL4 channel versus a cell count histogram plot. Results are reported as the mean ± SD of three separate experiments.
Prior to cell seeding, five sterile glass slides (Bellco Glass Inc., Vineland, NJ, USA) (0.05 mm in diameter) were inserted into each well of a non-adherent 6-well plate (Sarstedt, Nümbrecht, Germany). Primary human gingival fibroblasts were then seeded at 2 × 105 cells/well in DMEM supplemented with 10% FBS. Immediately after seeding, the cells were incubated with different concentrations (0, 10, 50, or 100 μg/mL) of peptide KSL-W in duplicate at 37 °C in a 5% CO2 incubator for 6 and 24 h, respectively. Following incubation, the cells were fixed with methanol (Fisher Scientific Co., Ottawa, ON, Canada) and glacial acetic acid (Merck KGaA) (75/25, v/v) for 15 min, followed by 3 washes with PBS. Thereafter, the fixed cells were incubated with 1 μg/mL Hoechst 33342 (H42) (Riedel de Haen, Seele, Germany) in PBS for 15 min at room temperature in the dark. After three washes with PBS, the samples were subjected to phase contrast microscopy and photographs were taken under an epifluorescence light microscope (Axiophot, Zeiss, Oberkochen, Germany). At least 10 fields from each slide were photographed and used to count the stained cells in each 2
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2.7. Cell migration/monolayer wound repair assay Primary human gingival fibroblasts were seeded (2 × 105/well) into 6-well plates and grown to confluence. A crossed scratch wound was created on each confluent monolayer by means of a 200 μL sterile pipette tip (PipetTipFinder, Lab Procurement Services, LLC, Knoxville, TN, USA) perpendicular to the bottom of the dish. This generated a wound approximately 0.44–0.50 mm in width [27]. The culture medium was then refreshed with new medium and the cells were incubated with or without peptide KSL-W at concentrations of 10, 50, or 100 μg/mL at 37 °C in a CO2 humid atmosphere. At the end of each incubation period (0, 12, 24, and 48 h), the control and peptideexposed cells were fixed with 4% paraformaldehyde solution (Alfa Aesar, Ward Hill, MA, USA) for 60 min at 4 °C and subjected thereafter to crystal violet staining (Sigma-Aldrich). Briefly, one milliliter of 1% w/v crystal violet solution in demineralized water was added to each well and the cultures were then incubated at room temperature for 15 min. The non-bound dye was removed from the wells by thorough washing with demineralized water, followed by drying at 37 °C. Digital photographs of each wound were taken (Coolpix 950, Nikon Canada) at various time points following the creation of the wound. Wound closure (cell migration) was investigated using the NIH Image J public domain image processing program to measure the distance between the opposite edges of the wound as a function of time. The distance between the wound edges was measured at least from 10 sites within the same wound. Data were presented as the percentage of initial wound (distance at time zero) using the following formula: [(distance at initial scratch − distance after individual treatment period) ÷ (distance at initial scratch)] × 100%. The KSL-W-treated and untreated cell cultures were compared, with the difference considered significant when p < 0.05. Results are reported as the mean ± SD of 5 separate experiments.
Fig. 1. Peptide KSL-W enhanced the early attachment of gingival fibroblasts. Cells were cultured with or without peptide KSL-W for 6 or 24 h and were then stained with Hoechst. The stained cells were counted and presented as the number of attached cells per microscope field. At least 10 fields from each slide were counted. P values were obtained by comparing the KSL-W-treated and untreated (Ctrl) values. Results are the mean + SD (n = 4).
Following collagen gel polymerization, 3 mL of culture medium with and without KSL-W (100 μg/mL) were added to each well, followed by incubation in a 5% CO2 humid atmosphere at 37 °C for various time periods. The contraction of each collagen gel specimen was measured by taking multiple measurements of the diameter. Results are reported as the mean ± SD of 4 separate experiments. 3. Effect of KSL-W on the expression of α-SMA by gingival fibroblasts Following culture in the presence or absence of KSL-W (100 μg/mL) for either 24 or 48 h, fibroblasts were detached from the culture plates. After 2 washes with PBS, cell lysates were prepared using a lysis buffer [25 mm of Tris–HCl, pH 8.0, 150 mm of NaCl, 1 mm of EDTA, 10% glycerol, 0.1% SDS, 0.05% sodium deoxycholate, 1% Triton X-100, and protease inhibitors (Sigma-Aldrich, cat. no. P2714) supplemented with anti-phosphatase cocktail III (Sigma-Aldrich, cat. no. P0044)]. The extracted protein concentrations were determined by the Bradford assay. Equal amounts of total protein (20–40 μg) in reducing sample buffer (61.5 mm of Tris, 100 mm of DTT, 2% SDS, and 10% glycerol) were boiled for 5 min and then electrophoresed by 10% SDS-PAGE. The proteins were transferred thereafter to PVDF membranes using a refrigerated Tris-glycine transfer buffer (25 mm of Tris, 192 mm of glycine, 100 μm of Na3VO4, and 20% methanol) for 1 h at 100 V. The blots were then incubated overnight at 4 °C with primary antibody against α-SMA (1∶100, clone 1A4, Sigma-Aldrich). β-actin (1:5000, A5441, Sigma-Aldrich), which exists in most cell type as component of the cytoskeleton, has been used as a housekeeping protein to correct for protein loading. Since both proteins (α-SMA and β-actin) have the same molecular weight (42 kDa), two membranes from the same samples with the same protein concentration were used to separately detect αSMA and β-actin. Finally, the membranes were washed and incubated for 1 h with the appropriate peroxidase-conjugated secondary antibodies. Detection was performed using the VersaDoc 5000MP Imaging System (Bio-Rad) and photographs were taken with Quantity One VersaDoc (Bio-Rad). Representative data out of three independent experiments were presented.
2.8. Effect of peptide KSL-W on the secretion of MMP-1, MMP-2, TIMP-1, and TIMP-2 by primary human gingival fibroblasts Supernatants were obtained from primary human gingival fibroblasts at 2 × 105/well in 6-well plates treated with 0, 10, 50, or 100 μg/mL of peptide KSL-W in duplicate for 3 and 6 days, respectively. MMP-1, MMP-2, TIMP-1, and TIMP-2 protein levels were analyzed by sandwich enzyme-linked immunosorbent assay (ELISA, R & D Systems, Minneapolis, MN, USA) using the colleced supernatants. Briefly, the cell culture media were collected in tubes containing 1 μL of a protease inhibitor cocktail (Sigma-Aldrich) for specific use with mammalian cell and tissue extracts. The culture media were then filtered through 0.22μm filters and used to quantify the MMP-1, MMP-2, TIMP-1, and TIMP2 concentrations, according to the manufacturer’s instructions. Culture supernatants were diluted 1/100 (MMP-1 and TIMP-1), 1/50 (MMP-2) and 1/10 (TIMP-2) using sample dilluent solution as recommended by the manufacturer. The plates were read at 450 nm and analyzed thereafter by means of a Model 680 Microplate Reader (Bio-Rad, Hercules, CA, USA). According to the manufacturer, the minimum detectable concentrations were under 1 pg/mL for MMP-1, 0.7 pg/mL for MMP-2, 3.5 pg/mL for TIMP-1, and 3.5 pg/mL for TIMP-2. Results are reported as the mean ± SD of 4 separate experiments. 2.9. Effect of peptide KSL-W on collagen gel contraction by gingival fibroblasts The effect of KSL-W on the ability of human gingival fibroblasts to contract collagen gel was determined using a previously described reconstituted type I collagen assay system [28]. Both the untreated and KSL-W-treated fibroblasts were used to measure the collagen gel contraction. Briefly, fibroblasts (3 × 105) were mixed with rat tail collagen I (Cat. No. A 10483-01, Gibco, Invitrogen, Burlington, ON, Canada) and then poured onto 35 mm-diameter tissue culture plates.
3.1. Statistical analysis Each experiment was performed at least three independent times, with experimental values expressed as means ± SD. The statistical significance of the differences between the control (absence of KSL-W) and the test (presence of KSL-W) values was determined by means of a 3
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Fig. 2. F-actin expression following cell treatment or not with KSL-W. Cells were cultured with or without various concentrations of KSL-W for 6 and 24 h and were then stained with phalloidin-FITC. Note the well-organized, dense filamentous actin cytoskeleton (arrows) in the KSL-W-treated cells versus the untreated ones. Representative images are from 3 different experiments. Scale bars = 100 μm.
4. Results
one-way ANOVA. Posteriori comparisons were done using Tukey’s method. Normality and variance assumptions were verified using the Shapiro-Wilk test and the Brown and Forsythe test, respectively. All of the assumptions were fulfilled. P values were declared significant at ≤0.05. Data were analyzed using the SAS version 8.2 statistical package (SAS Institute Inc., Cary, NC, USA).
4.1. Peptide KSL-W promoted primary human gingival fibroblast attachment, F-actin filament expression, and cell growth Primary human gingival fibroblasts were treated with various concentrations of peptide KSL-W and subsequently analyzed to determine the effect of this antimicrobial peptide on their attachment and morphology. As shown in Fig. 1, our quantitative evaluation (numbering) of the attached cells revealed a higher cell number in the presence of peptide KSL-W than in the untreated control. We observe a 4
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over 7% of the cells were in the S phase, and close to 14% were in the G2/M phase (Fig. 4). However, following exposure to KSL-W, the G0/G1 phase values significantly (p < 0.05) decreased, and markedly so in the presence of 50 and 100 μg/mL of the peptide. In contrast, the S and G2/M phase values considerably (p < 0.05) increased in the presence of 100 μg/mL of KSL-W. Similar results were obtained with fibroblasts stimulated with KSL-W for 48 h (Fig. 4). 4.3. Peptide KSL-W increased MMP-1, MMP-2, TIMP-1, and TIMP-2 secretion by human gingival fibroblasts Results of the protein analyses of the spent culture media reveal that peptide KSL-W modulated the levels of remodeling enzyme proteins MMP-1 and MMP-2, as secreted by the gingival fibroblasts (Fig. 5). Indeed, increased levels of remodeling enzymes were observed after 3 and 6 days of peptide treatment, compared to that observed in the untreated cultures. Significant (p < 0.05) increases in MMP-1 levels were observed in the spent culture media from cells treated with 50 and 100 μg/mL of KSL-W for 3 days. Similar findings were recorded for the 6-day fibroblast culture treated with the same amounts of peptide. The levels of secreted MMP-1 were greater after 6 days than after 3 days, regardless of the test conditions (control or KSL-W-treated cultures). Comparable data were obtained for the MMP-2 protein (Fig. 5), as the recorded levels secreted from cells treated with 50 and 100 μg/mL of KSL-W for 3 and 6 days were significantly higher (p < 0.05). Because peptide KSL-W increased MMP-1 and MMP-2 secretion, we also investigated the peptide’s effect on TIMP-1 and TIMP2, which are involved in regulating MMP activities. Fig. 6A shows that peptide KSLW increased TIMP-1 secretion by the primary gingival fibroblasts. This increase was statistically significant (p < 0.05) in the presence of both 50 and 100 μg/mL of KSL-W and at both 3 and 6 days of stimulation with the peptide. We also analyzed the effect of KSL-W on TIMP-2 secretion (Fig. 6B). An increase in TIMP-2 secretion was recorded in the cells treated with all three tested concentrations of KSL-W only at day 6 (Fig. 6B).
Fig. 3. Peptide KSL-W increased gingival fibroblast growth. Fibroblasts were treated with or without various concentrations of peptide KSL-W for 3 and 6 days. Viable cells were analyzed by trypan blue exclusion assay. The numbers of live cells were plotted as the mean + SD (n = 5). P values were obtained by comparing the KSL-W-treated and untreated values. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
significant (p < 0.01) increase of cell adhesion with 50 and 100 μg/ mL of KSL-W at 6 h of culture. The attached cell per microscope field goes from 25 ± 2 cells with the control to 38 ± 8 with 50 μg/mL of KSL-W, and 45 ± 5 cells with 100 μg/mL of the peptide. Similar observation was found in the 24 h culture, showing a significant (p < 0.01) increase of attached cells. The cell number per microscope field increased from 50 ± 2 cells with the control to 70 ± 6 with 50 μg/mL of KSL-W, and 81 ± 9 cells with 100 μg/mL of the peptide (Fig. 1). Cell attachment was further confirmed by F-actin cytoskeleton staining. Fig. 2 shows attached cells expressing dense F-actin staining at as early as 6 h of culture. The staining intensity of the F-actin filaments was also stronger in the KSL-W-treated cells than in the untreated culture (Fig. 2, arrows). These observations were confirmed after 24 h of culture, with cells expressing a well-organized and dense filamentous F-actin cytoskeleton (Fig. 2, arrows) in the KSL-W-treated culture, compared to the untreated culture. As shown in Fig. 3, compared to the unstimulated primary human gingival fibroblast culture, our cell growth analysis revealed a trend showing a slight increase in the number of viable primary human gingival fibroblasts after 3 days, although this increase was not statistically significant. Compared to the control, however, the KSL-W (10 and 100 μg)-treated human fibroblasts recorded a much higher number of viable cells after 6 days, and this increased growth was statistically (p < 0.01) significant. At low concentration (10 μg/mL) the peptide KSL-W has no effect on gingival fibroblast growth. This increase in cell growth was also maintained in the presence of 50 or 100 μg/mL of KSL-W. Overall results show that peptide KSL-W promoted the growth of primary human gingival fibroblasts after 6 days.
4.4. Peptide KSL-W promoted cell migration and wound closure Because peptide KSL-W was found to promote fibroblast attachment and growth, we investigated its modulatory effect on fibroblast migration. Our results show that the fibroblasts treated with peptide KSL-W actively migrated from both edges and after 48 h had closed the entire wound (Fig. 7). This healing rate was much more rapid than that observed with the untreated cells which showed 25–30% less closure after 48 h (Fig. 7). Furthermore, a significant (p < 0.05) reduction of wound distance after 12 and 24 h was observed in the cells treated with 50 and 100 μg/mL of peptide KSL-W versus the untreated controls (Fig. 7). It appears that the increase in fibroblast migration was associated with higher peptide concentrations. 4.5. Peptide KSL-W promoted collagen gel contraction Collagen gel assay is one of several well-established in vitro assays to investigate cell behavior and three-dimensional (3D) matrix remodeling that closely resembles cell behavior in vivo. To establish whether KSL-W was beneficial to tissue repair by boosting the contractile capacity of fibroblasts, we investigated the collagen gel contraction in KSL-Wstimulated fibroblasts. As shown in Fig. 8, the gel contraction was greater in the presence of peptide KSL-W than in the control (absence of KSL-W stimulation). Our quantitative results show that from an initial dimeter of 5.5 cm, the collagen gel has contracted to 2.8 ± 0.05 cm diameter in the control and 2.3 ± 0.01 cm in the gel being treated with 100 μg/mL of KSL-W after 24 h (Fig. 8). After 48 h, the collagen gel diameter was 2.1 ± 0.17 cm in the control group and 1.6 ± 0.04 cm in the 100 μg/mL of KSL-W (Fig. 8). Overall data confirm that KSL-W augmented the contractile activity of the fibro-
4.2. Peptide KSL-W modulated the cell cycle progression of human gingival fibroblasts We speculated that the KSL-W-induced increase in cell growth possibly occurred through a modulation of the cell cycle progression. To monitor the effect of peptide KSL-W on the cell cycle, gingival fibroblasts were treated with various concentrations of KSL-W for 24 or 48 h and the number of cells were quantified at different cell cycle phases (Fig. 4). Thus in the KSL-W-untreated group after 24 h of culture, approximately 70% of the cells were in the G0/G1 phase, while 5
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Fig. 4. Peptide KSL-W promoted cell division. Following stimulation for 24 and 48 h, the cells were detached and used for cell cycle testing by PI staining. Quantitation of the cell percentages at the different phases was determined by FAC analysis. P < 0.05 was obtained by comparing the KSL-W-treated and untreated cells (n = 3).
blasts, which may be explained by the expression of contractile fibers, such as α-SMA fibers. Compared to the untreated control cells, as demonstrated by Western blot analyses, the KSL-W-treated fibroblasts showed an increased expression of α-SMA contractile fibers (a stronger intensity of the protein band corresponding to α-SMA) following both 24 and 48 h of exposure to KSL-W (100 μg/mL) (Fig. 9).
cationic surface-active agent [31], peptide KSL-W has been shown to significantly reduce oral biofilm growth in vitro [32]. In addition to the inherent ability of antimicrobial peptides to kill bacteria, some of these peptides, show wound healing properties [33]. LL37, for example, at low concentration (5 μg/mL) stimulated airway epithelial cell proliferation and wound closure, while it induced necrosis at high concentration (> 20 μg/mL) [34]. We therefore sought to study the effect of peptide KSL-W on primary human gingival fibroblasts which are among the important wound healing cell types. Our results indicate that this antimicrobial peptide was non-toxic, even at a higher concentration (100 μg/mL), not like LL-37 [34], and was able to stimulate fibroblast growth. Nisin Z, an antimicrobial peptide [35] has been shown to enhance fibroblast defenses against infection [28]. Similarly, other antimicrobial peptides, in addition to their inherent antimicrobial activity, have been shown to stimulate the growth and migration of HaCat cells and human dermal fibroblasts
5. Discussion Various antimicrobial peptides are known to display in vitro activity against a wide range of bacterial pathogens and are thus being proposed as alternatives to develop novel antibacterial disease-control strategies [3,29]. Among available antimicrobial peptides, peptide KSL-W has demonstrated improved stability in simulated oral conditions with broad spectrum antimicrobial activity [30]. Furthermore, combined with sub-inhibitory concentrations of benzalkonium chloride, a known 6
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Fig. 6. Peptide KSL-W enhanced metalloproteinase inhibitor (TIMP-1 and TIMP-2) secretion. Note the increased levels of TIMPs with the high doses of peptide KSL-W after 6 days of stimulation. P < 0.05 was obtained by comparing the KSL-W-treated and untreated cells and the levels after 3 and 6 days (n = 4).
Fig. 5. Peptide KSL-W increased metalloproteinase (MMP-1 and MMP-2) secretion. Fibroblasts were stimulated for 3 and 6 days with different concentrations of KSL-W. Cell supernatants were used to measure the MMPs by specific ELISA. Note the increased MMP levels with the high doses of peptide KSL-W. P < 0.05 was obtained by comparing the KSL-W-treated and untreated (Ctrl) cells and the levels after 3 and 6 days (n = 4).
levels by human gingival fibroblasts. Similar observations were reported with α-defensin-1, shown to cause increased levels of mRNA expression of IL-6, IL-8, MMP-1, and MMP-3 by fibroblast-like synoviocytes [40]. In our study, when gingival fibroblasts were exposed to peptide KSL-W, the increased MMP secretion was paralleled by TMIP-1 and TIMP-2 secretion. Overall, peptide KSL-W stimulation enhanced fibroblast growth and increased MMP and TIMP secretion, which suggests that this peptide may contribute to cell migration and wound healing. To test this hypothesis, KSL-W-stimulated gingival fibroblasts were subjected to an in vitro wound scratch assay which showed that peptide KSL-W at 50 and 100 μg/mL promoted fibroblast migration and wound closure. These findings are in agreement with those of earlier studies showing that naturel antimicrobial peptides, such as human β-defensins-2, promoted cell migration and proliferation [41] and significantly accelerated wound closure when topically applied in a porcine model of infected skin wounds [42]. Studies have suggested that antimicrobial peptides on wounds serve as a modulator to help cells migrate during the wound healing process [43]. Essential molecules, such as epidermal growth factor receptor, STAT3 protein, and mitogen-activated protein (MAP)-kinases, have been shown to be involved in antimicrobial peptide-induced cellular reactions [43]. Because of the participation of these signaling molecules associated with cell activation and differentiation, we hypothesized that KSL-Wtreated fibroblasts may be involved in wound closure through their activation/differentiation into myofibroblasts. These cells are key players in the wound healing process and are responsible for cell-
[36]. Another study evidenced that ocellatin peptides extracted from frog Leptodactylus pustulatus skin controlled microbial growth with no adverse effect on human erythrocytes and a murine fibroblast cell line [37]. In our study, the effect of peptide KSL-W on fibroblast growth appears to have occurred through its influence on the cell cycle, as the KSL-W-treated cell cultures recorded a greater number of cells at the S and G2/M phases. As for the molecular target(s) of peptide KSL-W contributing to increased fibroblast growth in the treated gingival fibroblasts, this remains unclear. This effect is comparable to that of naturally occurring peptide catestatin secreted by human keratinocytes. Hoq et al. [38] showed that in addition to inhibiting the growth of pathogens, catestatin peptides promoted keratinocyte proliferation. Because fibroblasts adhered and proliferated in the presence of KSLW, this may thus have increased their physiological activity by stimulating their secretion of both MMP secretion inhibitors (TIMPs), as observed in our study. Similar to what has been reported with β-defensin-3 [39], the increased secretion of MMP-1 and MMP-2 by the KSL-W-treated fibroblasts may point to the involvement of peptide KSL-W in mediating part of the tissue remodeling processes. Alternatively, changes in MMPs could be considered as an inflammatory response by fibroblasts against the presence of peptide KSL-W, as was demonstrated [39] with other antimicrobial peptides, such as human β-defensin-3, which was shown to cause an increase in prostaglandin-(PGE) 2 and MMP-1 secretion 7
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Fig. 7. Peptide KSL-W increased gingival fibroblast migration/repair. Cells were cultured up to 100% confluence. Scratches were then made on each monolayer and the medium was refreshed and immediately treated with or without peptide KSL-W at different concentrations. The cultures were maintained for various time periods prior to observation and determination of the wound recovery. (A) Representative Photos 12 and 48 h post-wound (bar 50 μm). (B) Values are expressed as the mean ± SD (n = 5). The KSL-W-treated and untreated cultures were compared, with the difference considered significant at (*) p < 0.05.
8
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Fig. 8. Gingival fibroblasts stimulated with KSL-W contracted the collagen gel matrix. Following inclusion into the collagen matrix, fibroblasts were cultured in the presence or absence of KSL-W (100 μg/mL). The diameter of each collagen gel was measured after 24 and 48 h. Representative photos show the action of the KSL-W-treated and untreated cells on collagen gel contraction. The diameters of the collagen gels are presented. Values are expressed as the mean ± SD (n = 5). The KSL-W-treated and untreated tissues were compared, with the difference considered significant at p < 0.05.
These findings concur with others showing the ability of antimicrobial peptide LLKKK18 to accelerate wound closure and simultaneously improve the quality of healing [45]. Wound contraction occurred by the expression of α-SMA, which is critical to wound contraction and the differentiation marker of myofiboblasts from fibroblasts [46–48]. Concordantly, the KSL-W-enhanced fibroblast migration and collagen gel contraction shown in our study can be explained by the significant expression of α-SMA by the fibroblasts following stimulation with the peptide (100 μg/mL). Our study suggests a role of antimicrobial peptide in wound healing mechanisms through α-SMA. Our data suggest that one possible way by which antimicrobial peptide KSL-W helps wound healing is by increasing α-SMA expression in fibroblasts. This finding is significant for clinical application, with antimicrobial peptides being used to not only control infection but also contribute to the wound healing process.
Fig. 9. KSL-W increased α-SMA protein expression by gingival fibroblasts. Following cell stimulation with KSL-W (100 μg/mL) or not, proteins were extracted and analyzed by Western blot. Results show greater α-SMA expression in the KSL-W-treated cells than in the untreated cells.
mediated matrix contraction [44]. Through the contraction of their actin cytoskeleton, myofibroblasts at the wound site are able to reduce the initial size of the wound, thereby contributing to tissue repair [44]. In light of this, our study demonstrated that KSL-W-stimulated fibroblasts were able to induce the contraction of collagen gel matrix.
6. Conclusion Our study shows that antimicrobial peptide KSL-W promoted 9
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human fibroblast growth by increasing the cell population in the S and G2/M phases of the cell cycle. KSL-W also promoted the secretion of matrix metalloproteinase and tissue inhibitors of metalloproteinase, TIMP1 and TIMP2. Furthermore, this antimicrobial peptide increased fibroblast migration and collagen gel contraction in vitro, which may be due to the α-SMA protein expression and contraction as the results of the fibroblast-myofibroblast transdifferentiation induced by the peptide. Increase in α-SMA protein expression was observed in the KSL-Wtreated fibroblasts. Overall data suggest that KSL-W may have wound healing properties.
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