Antifibrotic Effects of Caffeine, Curcumin and Pirfenidone in Primary Human Keratocytes

We evaluated the small molecules (AFM) caffeine, curcumin and pirfenidone to find non-toxic concentrations reducing the transformation of activated human corneal stromal keratocytes (aCSK) to scar-inducing myofibroblasts (MYO-SF). CSK were isolated from 16 human corneas unsuitable for transplantation and expanded for three passages in control medium (0.5% FBS). Then, aCSK were exposed to concentrations of caffeine of 0–500 μM, curcumin of 0–200 μM, pirfenidone of 0–2.2 nM and the profibrotic cytokine TGF-β1 (10 ng/mL) for 48 h. Alterations in viability and gene expression were evaluated by cell viability staining (FDA/PI), real-time polymerase chain reaction (RT-PCR) and immunocytochemistry. We found that all AFMs reduced cell counts at high concentrations. The highest concentrations with no toxic effect were 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone. The addition of TGF-β1 to the control medium effectively transformed aCSK into myofibroblasts (MYO-SF), indicated by a 10-fold increase in α-smooth muscle actin (SMA) expression, a 39% decrease in lumican (LUM) expression and a 98% decrease in ALDH3A1 expression (p < 0.001). The concentrations of 100 µM of caffeine, 20/50 µM of curcumin and 1.1 nM of pirfenidone each significantly reduced SMA expression under TGF-β1 stimulation (p ≤ 0.024). LUM and ALDH3A1 expression remained low under TGF-β1 stimulation, independently of AFM supplementation. Immunocytochemistry showed that 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone reduce the conversion rate of aCSK to SMA+ MYO-SF. In conclusion, in aCSK, 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone significantly reduced SMA expression and MYO-SF conversion under TGF-β1 stimulation, with no influence on cell counts. However, the AFMs were unable to protect aCSK from characteristic marker loss.


Introduction
Corneal stromal scarring remains a major cause of blindness worldwide, with limited treatment options [1,2]. Damage to the mammalian cornea causes a rapid cellular reaction, which attempts to heal the wound. Disruption of the epithelial layer and basement membrane and injury to corneal stromal keratocytes (CSK) release a range of cytokines, including the platelet-derived growth factor (PDGF) and transforming growth factor β (TGF-β), the latter of which is probably the strongest pro-fibrotic agent [3,4].

Cell Number Analysis
To evaluate the potential toxic effects of the AFMs, aCSK were incubated in different media (Table 1) for 48 h, then stained using 5% fluorescein diacetate (FDA) and 5% propidium iodide (PI) and viable green cells were counted and compared. Table 1. Cell count and real-time polymerase chain reaction (RT-PCR) results for aldehyde dehydrogenase family 3 member A1 (ALDH3A1), lumican (LUM) and α-smooth muscle actin (SMA) in activated corneal stromal keratocytes (aCSK) after incubation for 48 h in media containing the indicated concentrations of the antifibrotic molecules (AFM) caffeine, curcumin and pirfenidone, with and without the profibrotic cytokine 10 ng/mL TGF-β1. Relative fold changes were calculated in comparison to the control group. Significant differences between groups are indicated by * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001. In curcumin, a significant cell reduction effect was seen at concentrations of 50 µM and above, in comparison to the control (p < 0.001; Table 1, Figures 1 and 2). aCSK cell numbers were further reduced at 100 µM of curcumin (cell count/mm 2 : 4.8 ± 3.0; p < 0.001) and 200 µM of curcumin (cell count/mm 2 : 0.0; p < 0.001).

SMA
In pirfenidone, the highest tested concentration of 2.2 nM (400 µg/mL) significantly reduced the aCSK cell count (p < 0.001; Table 1, Figure 1), in comparison to the control group.
The addition of 10 ng/mL of TGF-β1 to the medium did not influence the cell count at different AFM concentrations (p > 0.6; Table 1).
Consequently, the highest concentrations with no toxic effect on aCSK after 48 h of incubation were 100 of µM caffeine, 20 of µM curcumin and 1.1 nM of (200 µg/mL) pirfenidone. reduced the aCSK cell count (p < 0.001; Table 1, Figure 1), in comparison to the control group.
The addition of 10 ng/mL of TGF-β1 to the medium did not influence the cell count at different AFM concentrations (p > 0.6; Table 1).
Consequently, the highest concentrations with no toxic effect on aCSK after 48 h of incubation were 100 of µ M caffeine, 20 of µ M curcumin and 1.1 nM of (200 µ g/mL) pirfenidone.

Effects of TGF-β1 on aCSK
TGF-β1 addition to the control medium led to a 10-fold increase in SMA expression (p < 0.001, Figure 3, Table 1), a 39% decrease in LUM expression (p < 0.001) and a 98% decrease in ALDH3A1 expression (p < 0.001) in aCSK after 48 h of incubation.
LUM levels of aCSK incubated in 50 and 100 µ M caffeine media did not significantly differ from control aCSK. Caffeine 500 µ M aCSK showed a significant decrease in LUM expression compared to aCSK incubated in control media (p = 0.001, Table 1, Figure 3). No significant differences in LUM expression were seen between aCSK incubated in 50, 100 or 500 µ M caffeine + TGF-β1 in comparison to control + TGF-β1 aCSK.

Effects of Curcumin on aCSK
A highly significant decrease in SMA expression was seen when comparing curcumin 20 µ M and 50 µ M + TGF-β1 to control + TGF-β1 (p = 0.009 and p = 0.002, Table 1, Figure 3). (ALDH3A1) in activated corneal stromal keratocytes (aCSK) after incubation for 48 h in media containing the indicated concentrations of the antifibrotic molecules (AFM) caffeine, curcumin and pirfenidone, with and without the profibrotic cytokine 10 ng/mL TGF-β1. Relative fold changes were calculated in comparison to the control group. Significant differences between groups are indicated by * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001.
ALDH3A1 expression levels did not differ between 20 or 50 µM curcumin aCSK, in comparison to control aCSK ( Table 1). All groups incubated in the media containing TGF-β1 had low to no ALDH3A1 expression, regardless of curcumin supplementation.

Effects of Pirfenidone on aCSK
The incubation of aCSK in pirfenidone 1.1 or 2.2 nM medium led to no significant difference in SMA expression compared to control aCSK. SMA expression levels were significantly lower in pirfenidone 1.1 nM + TGF-β1 compared to control + TGF-β1 (p = 0.024, Table 1, Figure 3). The SMA expression in pirfenidone 2.2 nM + TGF-β1 did not differ from control + TGF-β1 (p = 0.1).
A significant decrease in ALDH3A1 expression was found when comparing pirfenidone 1.1 or 2.2 nM to control (p < 0.001 and p < 0.009, Table 1, Figure 3). All groups incubated in media containing TGF-β1 showed low to no ALDH3A1 expression, regardless of pirfenidone supplementation.

Immunocytochemistry
Immunocytochemistry staining showed no SMA expression in control aCSK ( Figure 4).

Discussion
In this study, we investigated the antifibrotic effect of caffeine, curcumin and pirfenidone on cultured primary human aCSK. We found that, in high concentrations, the AFMs exerted cytotoxic effects. The highest concentrations with no toxic effect on aCSK after 48 h of incubation were 100 µ M of caffeine, 20 µ M of curcumin and 1.1 nM (200

Discussion
In this study, we investigated the antifibrotic effect of caffeine, curcumin and pirfenidone on cultured primary human aCSK. We found that, in high concentrations, the AFMs exerted cytotoxic effects. The highest concentrations with no toxic effect on aCSK after 48 h of incubation were 100 µM of caffeine, 20 µM of curcumin and 1.1 nM (200 µg/mL) of pirfenidone. TGF-β1 addition to the control medium induced the conversion of aCSK to SMA + MYO-SF and reduced the expression of the typical CSK markers lumican and ALDH3A1. The concentrations of 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone each significantly reduced SMA expression and MYO-SF conversion under TGF-β1 stimulation with no influence on cell counts. However, they were unable to protect from CSK marker loss.
Stromal cellular responses such as CSK activation and apoptosis after surgical trauma have been studied in the corneas of mice [24], rats [5], rabbits [25] and cats [26], but rarely, due to the scarcity of tissue, in human corneas [27], where the responses may be attenuated just as the endothelial response to injury is [28]. In particular, CSK activation and regeneration appear to be decreased in human corneas after photorefractive keratectomy compared with, e.g., the responses in rabbits [29].
Hence, investigating the effect of caffeine, curcumin and pirfenidone on primary human aCSK is crucial in the search for a means of preventing haze and MYO-SF differentiation, while stimulating the regeneration of the corneal stroma and epithelium, and preserving or restoring normal ocular optics.
Caffeine (1,3,7-tri-methylxanthine) is one of the most consumed food additives worldwide and has wide-ranging pharmacological activities including effects on the central nervous, cardiovascular and respiratory systems [21]. It can act as an antagonist of adenosine receptors, an inhibitor of phosphodiesterases and an activator of ryanodine receptors. Caffeine is similar in structure and function to theophylline and can improve lung function in asthmatics by inducing bronchodilation [30]. In recent years, caffeine has been shown to exhibit anti-fibrotic effects in the liver. The consumption of caffeine, often in the form of coffee, is associated with reduced hepatic fibrosis in patients suffering from chronic hepatitis C virus infection [31]. In in vivo animal models of liver fibrosis, caffeine can reduce collagen deposition and collagen mRNA [32,33], and can block the expression of the profibrotic cytokine TGF-β [34]. Furthermore, caffeine can inhibit profibrotic responses in hepatic stellate cells, the key effector cell in the development of liver fibrosis [35]. In the lung, caffeine appears to exhibit its anti-fibrotic effects through distinct actions on both epithelial cells and fibroblasts, which are two of the key effector cells involved in the pathogenesis of pulmonary fibrosis. It has previously been reported that caffeine is capable of interrupting TGF-β-induced Smad signaling in a lung epithelial cancer cell line [36]. However, Tatler et al. recently showed that caffeine also abrogated profibrotic responses to TGF-β in lung fibroblasts. It inhibited basal expression of the SMA gene and reduced TGF-β-induced increases in profibrotic genes [21].
In our study, we showed, for the first time, that 100 µM of caffeine significantly reduced SMA expression and MYO-SF conversion in primary human aCSK. However, caffeine was unable to protect from CSK marker loss.
Caffeine is not known to undergo a significant first-pass metabolism and generally reaches its peak plasma concentrations within 30-120 min of its administration. Studies have shown that serum caffeine levels from moderate coffee consumption usually range between 20 and 50 µmol/L and do not tend to exceed levels of 70 µmol/L [37]. In general, it has been noted that toxicological symptoms often begin above concentrations of 15 mg/L (≈7.7 µM, generally mild psychological side effects such as irritability and nervousness, but also potentially palpitations, nausea, tremor, perspiration and paresthesia), while a concentration of 50 mg/L (≈257 µM) is considered "toxic". Caffeine concentrations of 80 mg/L (≈411 µM) or greater are considered lethal [38] and, accordingly, 500 µM of caffeine reduced CSK counts and did not exert an antifibrotic effect in our setup.
Consequently, the topical, rather than systemic, application of 100 µM of caffeine eye drops in a corneal scarring model could be a coherent next investigative step.
Fibrosis within the cornea tends to be dynamic, with ongoing MYO-SF generation and apoptosis. Once mature MYO-SF develop, they persist until the requisite source of TGF-β1 and/or TGF-β2 to maintain viability is sufficiently reduced by repair. Once the levels of these TGF-β isotypes drop in the stroma, then IL-1 produced by surrounding cells (CSK, SF and MYO-SF (autocrine)), unopposed by TGF-β1 or TGF-β2, triggers increased apoptosis of the MYO-SF [39]. In our experimental setup, the strong stimulation by 10 ng/mL of TGF-β1 could have protected MYO-SF from the toxic effects of 500 µM of caffeine and, consequently, increased overall SMA expression.
Interestingly, the antifibrotic caffeine did not protect aCSK from the loss of their characteristic markers (i.e., lumican, ALDH3A1) during stimulation by 10 ng/mL of TGF-β1. The fact that CSK marker expression and SMA + MYO-SF conversion show different responses to fibrosis inhibition has been shown before.
Seidelmann et al. demonstrated that the expression of the MYO-SF marker SMA decreased with incremental human platelet lysate (hPL) substitution in CSK and SF, implying an antifibrotic effect, which they attributed to the basic fibroblast growth factor (bFGF) and hepatocyte growth factor (HGF) found in hPL. Nevertheless, CSK markers decreased with higher hPL substitution [13].
Similarly, Jester et al. incubated primary rabbit CSK with 10 ng/mL of bFGF and found a fibroblast-like phenotype but negative SMA immunocytochemistry after 7 days of culture [40].
Curcumin (diferuloylmethane), derived from the rhizome of Curcuma longa L., belongs to the polyphenols. It was isolated over 140 years ago by Vogel and was synthesized in 1913 by Lampe [41]. Curcumin has been used for thousands of years in traditional Chinese medicine and Ayurvedic medicine in Asian countries as an active ingredient of herbal remedies to treat liver diseases, rheumatoid diseases, diabetes, atherosclerosis, infectious diseases and cancer [42]. It is characterized by an anti-oxidant, anti-inflammatory, antimutagenic, anti-microbial and anti-cancer activity [41]. Oral curcumin substitution was shown to decrease serum concentrations of TNFα, IL-6, TGF-β and MCP-1, major mediators of inflammation in many diseases [43].
The inhibitory effect of curcumin was also described for epidermal mouse keratinocytes [46]. Curcumin diminished the uPA levels induced by TGF-β1 in immortalized skin keratinocytes and TGF-β-induced synthesis of fibronectin, as well as inhibited TGF-β-stimulated cell migration and invasiveness [46]. In a rabbit in vivo model of suturing-induced corneal neovascularization, Kim et al. demonstrated the efficacy of topical curcumin in inhibiting angiogenesis through decreasing VEGF mRNA levels and NF-κB phosphorylation [47].
Curcumin at concentrations of 5-20 µmol/L significantly inhibited UVB-induced secretion of IL-6 and IL-8 by human limbus epithelial cells in a dose-dependent manner, while curcumin alone did not affect the secretion of IL-6 and IL-8 [22]. The upregulation of NF-κB and MAPK pathways induced by UVB treatment was significantly inhibited by curcumin, suggesting that the NF-κB and MAPK pathways are involved in the inhibitory effect of curcumin in the UVB-induced production of IL-6 and IL-8 [22]. Consequently, curcumin inhibits numerous pathways that are also involved in corneal scarring, e.g., TGF-β and MAPK.
In our study, we showed, for the first time, that 20 µM of curcumin significantly reduced SMA expression and MYO-SF conversion in primary human aCSK. However, curcumin was unable to protect from CSK marker loss.
Bolger et al. examined the pharmacokinetics following the intravenous infusion of liposomal curcumin. Healthy individuals showed a curcumin peak plasma concentration of about 2000 ng/mL (≈5.4 µM) after 2 h at a dose of 240 mg/m 2 and of nearly 3000 ng/mL (≈8.1 µM) after 2 h [48]. In a dose-escalation and pharmacokinetic study of nanoparticle curcumin with six healthy human volunteers, Kanai et al. measured a peak plasma concentration of 275 ± 67 ng/mL (≈0.8 µM) approximately 2 h after receiving a single dose of 210 mg [49].
Several trials on healthy subjects have supported the safety and efficacy of curcumin [50]. Despite this well-established safety, some negative side effects have been reported. Seven subjects receiving 500-12,000 mg in a dose-response study and followed for 72 h experienced diarrhea, headache, rash, and yellow stool [51]. In another study, some subjects receiving 0.45 to 3.6 g/day of curcumin for one to four months reported nausea and diarrhea and an increase in serum alkaline phosphatase and lactate dehydrogenase contents [52].
In vitro data indicated a toxic effect of 15 µM of curcumin microemulsion in a hepatocellular HepG2 cell line [53].
Accordingly, we found that curcumin in concentrations higher than 20 µM showed substantial toxic effects, with hardly any aCSK left after 48 h incubation in 100 and 200 µM. The reduced lumican expression in curcumin 50 µM aCSK is most likely also an indication for toxicity.
To target corneal scarring, further investigations with topical curcumin eye drops at concentrations of ≤20 µM could be a safe approach.
In the eye, pirfenidone has been shown to inhibit the activity of human Tenon's fibroblasts in vitro [64], and has also been tested as an adjunctive postoperative antifibrotic for strabismus surgery in rabbits [65]. In addition, it has been evaluated as a postoperative anti-scarring agent for use in glaucoma surgery in a lagomorph model, and it was recently reported to successfully inhibit TGF-β1-induced equine corneal fibrosis in vitro [66,67]. In mice, Singh et al. showed that the inhibitory effects of pirfenidone on TGF-β and PDGF are efficacious in decreasing myofibroblast formation from both keratocyte and bone marrow-derived precursors [68].
Pirfenidone nanoparticles also improved corneal wound healing and prevented corneal fibrosis in Sprague Dawley rats in vitro and in vivo [69]. In addition, stromalwounded ex vivo canine corneas exhibited greater optical clarity when treated with pirfenidone than when placebo treated at 21 days [20].
In our study, we showed, for the first time, that 1.1 nM (200 µg/mL) of pirfenidone significantly reduced SMA expression and MYO-SF conversion in primary human aCSK. However, pirfenidone was unable to protect from CSK marker loss under TGF-β stimulation.
Pirfenidone is commercialized as an oral immediate release formulation with an administration of 801 mg/day during the first week, followed by an increase to 1602 mg/day during the second week and a subsequent dose increase to reach 2403 mg/day after 15 days of treatment. Pirfenidone use, however, is limited by the occurrence of adverse events [70]. Gastrointestinal reactions, such as nausea, dyspepsia, diarrhea, abdominal discomfort and vomiting, are frequently associated with pirfenidone administration. Anorexia, fatigue, sedation and photosensitivity have also been reported. The frequency and intensity of these responses appear to decrease with time. However, adverse events often lead to dose reductions or treatment withdrawal [71].
In a pharmacokinetic study on 28 patients with idiopathic pulmonary fibrosis and chronic hypersensitivity pneumonitis, pirfenidone showed a rapid absorption rate, reaching the maximal concentration at around 0.86 ± 0.37 h and a peak concentration of 19.81 µg/mL [71].
Jiang et al. previously demonstrated that pirfenidone inhibited human umbilical vein endothelial cell (HUVEC) viability at concentrations of ≥300 µg/mL, but eye drops containing 1000 µg/mL of pirfenidone administrated four times per day for 14 days reduced corneal edema, promoted corneal wound healing and inhibited neovascularization formation in an alkali burn rat model [23]. Consequently, to target corneal scarring, topical pirfenidone eye drop treatment appears to be the safest approach.
Interestingly, aCSK incubated in media containing 1.1 or 2.2 nM (200 or 400 µg/mL) of pirfenidone showed a significant increase in lumican expression, which could indicate a protective effect against CSK marker loss during 0.5% FBS stimulation [4]. However, the ALDH3A1 expression changed in the opposite direction, which implies more complex effects of pirfenidone on the CSK cell character.
As a limitation, in this in vitro cell culture pilot study, we investigated the antifibrotic effects of the AFMs through alterations in CSK marker and SMA expression. Nevertheless, corneal fibrosis is a complex dynamic process influenced by various genes and proteins [72,73], among them, interleukin 1 and 6, matrix metallopeptidases, collagens, fibronectin, PDGF, HGF and keratinocyte growth factor (KGF). However, the TGF-β1and 2-dependent induction of SMA + MYO-SF is an integral part of organ fibrosis, and is frequently used to screen for the antifibrotic effects of different components in the cornea and other organs in and ex vivo [21,23]. Further studies using, e.g., proteomics or genomics at different time points, could help us to better understand the specific effects of the SMA-suppressive AFMs found in this study on corneal and CSK fibrosis induction.
To summarize, we found that 100 µM of caffeine, 20 µM of curcumin and 1.1 nM of pirfenidone each significantly reduced SMA expression and MYO-SF conversion under TGF-β1 stimulation, with no influence on cell counts. However, they were unable to protect from CSK marker loss. Further studies are necessary to investigate the ideal concentrations and application modes of caffeine, curcumin and pirfenidone for the inhibition of corneal scarring, as well as their potential for CSK ex vivo expansion for tissue-engineering and research purposes.

Cell Culture of aCSK
The CSK were cultured in aCSK basal medium containing 0.5% FBS until passage 3. After 24 h, the medium was exchanged for new medium containing the according substitutes ( Table 2). The basal medium group served as the control. After 48 h of culture in the according medium, the cells were harvested for further testing.

Cell Number Analysis
The cells were seeded at 9000 cells/1.8 cm 2 on collagen-I-coated 24-well plates (Corning, New York, NY, USA) and incubated in different media (Table 2) for 48 h at 37 • C. Then, the media were removed and 5% fluorescein diacetate (FDA) and 5% propidium iodide (PI) in PBS (both from Sigma-Aldrich) were added for live/dead staining. Samples were imaged by fluorescence microscopy (Leica DM6000B microscope, Leica Microsystems GmbH, Wetzlar, Germany). The numbers of live (green fluorescence) and dead cells (red fluorescence) were quantified in 10 random fields per well, using the cell counter plugin for Image J (version 1.53o, Wayne Rasband, Bethesda, MD, USA) [75]. Experiments were conducted in triplicate for 6 donors.

Immunocytochemistry
The cells were seeded at 9000 cells/1.8 cm 2 on collagen-I-coated glass cover slips (VWR International, Radnor, PA, USA). After 48 h of culture in different media (Table 2), the cells were fixed with neutral buffered 4% paraformaldehyde (Sigma-Aldrich). After quenching with 50 mM of ice-cold ammonium chloride (Sigma-Aldrich), the samples were washed with PBS containing 0.2% bovine serum albumin (BSA, Sigma-Aldrich) and blocked with 1% bovine serum albumin and Triton X (1 µL/mL, Sigma-Aldrich), followed by incubation with the primary antibody mouse anti-SMA1 (1:200, Invitrogen) for 2 h at room temperature. After these buffer washes, the samples were incubated with the respective secondary antibodies conjugated with Alexa Fluor 555 (donkey anti-mouse, 1:2000, Invitrogen) for 1 h. The samples were buffer-washed, mounted with Prolong Gold antifader reagent with DAPI (Invitrogen) for nuclear contrast staining and visualized by fluorescence microscopy (Leica DM6000B microscope, Leica Microsystems GmbH) and Diskus Viewer 4.8 (Hilgers Technisches Büro e. K., Königswinter, Germany). Experiments were conducted in triplicate for 5 donors.

Real-Time Polymerase Chain Reaction (RT-PCR)
The cells were seeded at 9000 cells/1.8 cm 2 on collagen-I-coated 24-well plates (Corning, New York, NY, USA) and incubated in different media ( Table 2) for 48 h at 37 • C. The total RNA from cultured cells was extracted using RNeasy MiniKit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. Reverse transcription was carried out with the Reverse Transcription System (Promega, Madison, WI, USA). Alterations in gene expression were analyzed by quantitative real-time PCR (RT-PCR) using the LightCycler FastStart DNA Master SYBR Green I kit (Roche) with the LightCycler 1.2 (Roche). The samples were taken in duplicate using the following primers (Supplementary Table S1): glyceraldehyde-3-phosphate dehydrogenase (GAPDH), α-smooth muscle actin (SMA), lumican (LUM) and aldehyde dehydrogenase family 3 member A1 (ALDH3A1). Relative fold changes in gene expression were analyzed using the comparative CT (2 −∆∆CT ) method for 6 different donors [76]. Relative fold changes were calculated in comparison to the control group.

Statistical Analysis
All data are expressed as mean ± standard deviation (SD). Statistical analyses were performed with SPSS version 22.0 (IBM, Chicago, IL, USA). The Mann-Whitney U test or Wilcoxon rank-sum test were used to compare cell numbers and gene ratios. A p value of ≤0.05 was considered statistically significant.

Institutional Review Board Statement:
The study was conducted in accordance with the Declarations of Helsinki and Istanbul. Ethical committee approval was obtained from the Ethics Committee at the RWTH Aachen Faculty of Medicine (EK 291/20, approval date 18 August 2020).

Informed Consent Statement:
Informed consent for cornea donation and scientific use in case of unsuitability for transplantation was obtained from next-of-kin.
Data Availability Statement: All data can be requested form the corresponding author.