Quiescent keratocytes fail to repair MMC induced DNA damage leading to the long-term inhibition of myofibroblast differentiation and wound healing

Cell culture studies

Rabbit corneal keratocytes were isolated from rabbit eyes (Pel Freez, Rogers, AR) and cultured under serum-free conditions as previously described [20,21]. Cells were plated at a density of 1–5×10⁴ cells/cm² in polystyrene coated tissue culture dishes (Falcon; Becton Dickinson, Franklin Lakes, NJ) and allowed to attach for 72 h. Cells were then treated with various concentrations of MMC (Calbiochem, La Jolla, CA) for 5 min and then rinsed in serum-free media. Cells were then cultured with and without transforming growth factor-β (TGFβ, 5 ng/ml; Sigma-Aldrich, St. Louis, MO) for various times and evaluated by immunocytochemistry, western blotting, and RT–PCR.

Immunohistochemistry

Cells were fixed in 2% paraformaldehyde in PBS (pH=7.4), then treated with acetone (−20 °C) for 3 min, rinsed with PBS, blocked with goat serum (dilution 1:10 in PBS; Cappel; MP Biomedicals, Solon, OH) for 30 min at 37 °C and stained with monoclonal antibodies to α-smooth muscle actin (αSMA; Sigma, St. Louis, MO) to measure myofibroblast differentiation, the nuclear cell cycle protein Ki67 (clone MIB-2; Inmunotech, Westbrook, ME) to measure cell cycle entry and γH2AX (Abcam, Cambridge, MA) to assess DNA damage [16-18]. Sections were then stained with FITC conjugated goat anti-mouse IgG (Southern Bioech, Birmingham, AL) and counterstained with Alexa 543 phalloidin and DAPI. Sections were then evaluated using the Nikon Eclipse E600 epifluorescence microscope (Nikon Instruments Inc., Melville, NY).

Cell proliferation

To assess the effects of MMC on TGFβ induced cell proliferation, keratocytes were cultured in 6-well plates (Falcon) and treated with various concentrations of MMC for 5 min. Cells were then cultured with and without TGFβ and the change in cell number measured as previously describe using a Leica DMIRB Inverted microscope (Leica Microsystems, Werzlar, Germany) and Optronics CCD camera (Goleta, CA) [22]. At least three wells/treament condition were measured and the experiment repeated 3 times.

Western blotting

Total proteins were solubilized in buffer containing 25 mM Tris-HCl (pH 7.4), 1 mM EDTA, 1 mM EGTA, 10 mM dithiothreitol, 1% sodium dodecyl sulfate (SDS), 5 μg/ml antipain, 5 μg/ml pepstatin A, and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Proteins were electrophoresed on precast 10% Tris-glycine sodium dodecyl sulfate polyacrylamide (SDS–PAGE) gels (Invitrogen, Carlsbad, CA). Proteins were transferred to a polyvinylidene difluoride membranes (Bio-Rad, Hercules,CA), blocked for 2 h with 5% BSA in Tris saline and probed with antibodies to αSMA (Sigma-Aldrich) at 4 °C overnight. Blots were then washed with Tween-20 and Tris-buffered saline (50mM Tris,150mM NaCl and 0.05% Tween-20) and incubated for 1 h with a Cy3 conjugated goat anti-mouse IgG (1:5000; Southern Biotech). Western blots were then scanned at an excitation of 532 nm and emission of 580 nm using a fluorescent imager (FMBIO III; Hitachi-MiraiBio Inc., Alameda, CA).

RNA extraction and PCR

Cells were washed 2× in cold PBS then directly lysed in RLT buffer (Qiagen, Valencia, CA) and the supernatant recovered. The samples were then passed over QiaShredder columns and then processed over RNeasy columns (both from Qiagen). The RNA was eluted from the silica column with 50 μl water, and the product was analyzed using a NanoDrop Spectrophotometer (Thermo Scientific, Wilmington, DE) to estimate RNA concentrations. The RNA (500 μg) obtained from cultured cells was converted to cDNA using the QuantiTect Reverse Transcription Kit (Qiagen). Reactions were performed for 30 min at 42 °C, and 5 min at 95 °C, followed by cooling to 4 °C. After synthesis, the cDNA from all sources was diluted to equivalent input RNA levels with TE (10 mM Tris/HCl, 1 mM EDTA [pH 7.5]; 10 ng input RNA/μl) and stored at −20 °C until use. cDNA samples were subjected to PCR using specific primers (Table 1). Primers were designed using Primer 3 Internet software program (The Whitehead Institute, Cambridge, MA), and their specificities were confirmed by a BLAST Internet software-assisted search of the non-redundant nucleotide sequence database (National Library of Medicine, Bethesda, MD). Polymerase chain reactions (PCR) were carried out as previously described with 5–25 ng reverse-transcribed RNA and 200 nM forward and reverse primers, in a total volume of 25 ml [23]. For quantitative real time PCR (qPCR), samples were normalized by b2-MG amplification and were amplified using the QuantiTect Syber Green reagents (Qiagen) in triplicate using an MJ Research Opticon Thermal Cycler (Waltham, MA).  PCR controls without reverse transcriptase (water control) or with normal human genomic DNA as a template were routinely negative. PCR controls without reverse transcriptase (water control) were routinely negative. Product specificities were confirmed by DNA sequencing the products.

Comet assay

DNA repair by corneal fibroblasts and keratocytes was assessed by Comet assay using the protocol previously described by Cordelli et al. [24]. Briefly, slides were prepared by dipping in 1% agarose/PBS and allowing to air dry to a thin film. Cells were harvested 24 h post MMC treatment by scraping in cold PBS, centrifuged at 500× g and the cell pellet re-suspended in PBS to 10⁷ cells/ ml. The cell suspension (10 μl) was quickly mixed with 65 μl 0.7% Low Melting Point agarose/PBS, coverslipped, and the agarose allowed to gel at 4 °C. After gelling, the coverslip was removed and the gel overlaid with an additional 100 μl of 0.7% LMP agarose/PBS and allowed to gel. Slides were then submerged overnight at 4 °C in lysis buffer containing: 2.5M NaCl, 100 mM EDTA, 10 mM Tris/HCl pH 10, 1% Triton X 100 and 10% DMSO. The following morning, the slides were placed in lysis buffer containing 10 mM DTT and allowed to incubate, for 30 min at 4 °C. The slides were then placed under freshly prepared electrophoresis buffer composed of: 300 mM NaOH, 1 mM EDTA, adjusted to pH 13 with HCl and held at 4 °C for 20 min. The samples were then electrophoresed at 4 °C, for 5 min at 25V. The slides were then immersed in 0.3 M sodium acetate in 70% alcohol for 30 min, then fixed in 100% alcohol for 2 h, followed by 70% alcohol for 5 min and then allowed to air dry. The slides were stained by immersion in 2 μg/ml Ethidium Bromide and photographed under a 20× objective. Slides were scored using CometScore software v1.5 (TriTek Corporation, Summerduck, VA).

Rabbit studies

New Zealand albino rabbits were used in this study and all procedures were approved by the UCI IACUC and conducted in accordance with ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Prior to injury, all rabbits were anesthetized with intramuscular injections of xylazine (5 mg/Kg) (Anased, Lloyd Laboratories, Shenandoah, IA) and ketamine (22 mg/Kg) (Bioniche Pharma USA LLC, Lake Forest, IL). Tetracaine hydrochloride 0.5% eye drops (Alcon Laboratories Inc., Forth Worth, TX) were instilled into each right eye, and a speculum was used to open the eyelids. After surgery, eyes received topical gentamicin sulfate (Akorn, Buffalo Grove, IL) three times a day for 3 days and intramuscular buprenorphine (Buprenx; 0.1 mg/kg; Reckitt Benckiser Healthcare, Hull, UK) once and as needed for relief of pain.

DNA damage study

A total of 6 rabbits were used in this study. An 8-mm trephine (Bausch & Lomb, Rochester, NY) was used to mark the central cornea and the epithelium was then removed using a Bard Parker® #11 surgical blade (Becton Dickinson AcuteCare, Franklin Lakes, NJ). Both eyes of each rabbit were then randomly assigned to four groups of 3 eyes each and were treated with either 0.2 mg/ml or 0.02 mg/ml MMC for 15 s or 60 s. Animals were allowed to recover and then sacrificed 3 h after treatment.

MMC and delayed injury study

A total of 10 rabbits were used in this study. Initially rabbits received a unilateral epithelial scrape injury and then randomly assigned to two group of 5 rabbits each and treated with 0.2 mg/ml MMC for 60 s or vehicle control applied using a surgical sponge as previous described [25]. Animals were allowed to recover for 8 weeks and then received a lamellar keratectomy (LK) injury in the same treated eye using techniques previously reported [26,27]. Rabbits were then followed by in vivo confocal microscopy and then sacrificed 3 months after LK injury.

In vivo confocal microscopy (CM)

Under anesthesia, animals received a baseline scan and were then followed at various times after injury by in vivo confocal microscopy (CM) to measure epithelial and stromal thickness and haze as described previously [28,29]. All in vivo CM examinations were performed using a tandem scanning confocal microscope (TSCM; Tandem Scanning Corporation, Reston, VA) with a 24× surface-contact objective (numerical aperture, 0.6; working distance, 1.5 mm). One drop of 2.5% hydromethylcellulose (Gonak, Akorn, Buffalo Grove, IL) was placed on the tip of the objective as a coupling gel. All camera settings were kept constant throughout the experiment. For each examination, repeated through focus data sets were obtained from the central corneal region. A total of three to five through-focus data sets for each eye at each time point were collected in the thinnest region of the cornea.

Ex vivo microscopy

After animals were sacrificed, corneas were fixed by anterior chamber perfusion for 4 min with 2% paraformaldehyde in phosphate-buffered saline, pH 7.4. After perfusion, corneas were dissected and left in 2% paraformaldehyde overnight. For assessment of DNA damage following MMC treatment, fixed corneal tissue was embedded in O.C.T Compound (Tissue-Teck, Sakura Finetek, Torrance, CA) and snap frozen in liquid nitrogen. Blocks were then cut on a Leica CM 1850 cryotome (Leica, Wetzlar, Germany) and stained with antibodies specific of γH2AX for 1 h followed by staining with goat anti-mouse IgG conjugated to FITC for 1 h. Slides were then counter stained with DAPI (300 nM, Invitrogen, Carlsbad, CA) and imaged using a Nikon Eclipse E600 epifluorescent microscope equipped with a Photometrics CoolSnap FX camera (Roper Scientific, Tucson, AZ) and images digitized using Meta Imaging Series software (Molecular Devices, Downington, PA). All images were taken using the same camera settings and exposure time, and all slides were stained on the same day. To quantify the intensity of γH2AX staining over keratocyte nuclei, the DAPI channel was used to define regions of interest over nuclei using the create region tool in the Meta Imaging software. These regions were then applied to the γH2AX channel, and the average pixel intensity over the nuclei measured and recorded. To adjust for differences in staining, the average background image intensity overlying non-nuclear areas was also measured and subtracted from the measured nuclear intensity for each slide. On average, a total of 20 nuclei from each corneal section from each region were measured.

To assess corneal fibrosis, collagen organization of the corneas was visualized by non linear optical imaging of second harmonic generated signals from collagen (SHG). SHG signals to detect collagen were generated using 800-nm infrared femtosecond laser pulses to obtain maximum SHG emission signal at 400 nm [25]. SHG forward-scattered signals passing through the tissue were collected using an 0.8 NA condenser lens with a narrow band-pass filter (400/50) placed in front of the transmission light detector. Backward-scattered SHG signals were detected using the Meta Detector set to collect 390 to 410 nm band width on the Zeiss 510 Meta Laser Scanning Confocal microscope (Carl Zeiss Microscopy GmbH, Jena, Germany). To assess cell density in the same optical plane, corneal blocks were also stained with Syto 59 (Invitrogen), and imaged using laser scanning confocal microscopy.

Statistical analysis

All results are reported as mean±standard deviation. Differences between treatment groups were assessed by two-way repeated-measures ANOVA and Bonferroni multiple comparisons (Sigma Stat version 3.11; Systat Software Inc., Point Richmond, CA). For the live rabbit studies we used a sample size of five eyes per group, with a power of 0.95 and α=0.05 to detect a 50% reduction in haze. All tissue culture experiments were performed in triplicate and repeated at least three times.

Effects of MMC on TGFβ-induced myofibroblast differentiation

To establish whether MMC blocked myofibroblast differentiation of quiescent keratocytes, cultures were stained with specific antibodies to αSMA, a known biomarker for myofibroblast differentiation [21], and the number of cells expressing αSMA quantified. By comparison, keratocytes cultured under serum-free conditions maintained a dendritic morphology and showed no staining for αSMA (Figure 1A), while cells exposed to TGFβ alone showed cell spreading, increased actin filament assembly and marked expression of αSMA (Figure 1B). When cells were pre-treated with MMC for 5 min and then immediately treated with TGFβ, MMC at concentrations of 0.02 mg/ml and 0.002 mg/ml blocked TGFβ induced cell spreading, actin filament assembly and expression of αSMA at 72 h (Figure 1C,1D). Concentrations above 0.02 mg/ml induced marked keratocyte cell death. Quantifying the number of cells expressing αSMA showed that the pre-treatment of cells with MMC dose dependently and significantly reduced (p<0.001) the number of cells staining for αSMA. Western blots also confirmed the reduction in expression of αSMA after 72 h of TGFβ treatment (Figure 1E, insert). Overall, these data showed that MMC leads to abnormal TGFβ-induced myofibroblast differentiation of rabbit corneal keratocytes.

Effects of MMC on TGFβ-induced cell replication and gene transcription

Since DNA damage induced by MMC is known to block cell replication and gene transcription, we next evaluated the effect of MMC on TGFβ-induced cell growth and gene expression of quiescent keratocytes (Figure 2). While keratocytes showed no increase in cell number over 7 days in culture with or without MMC treatment (data not shown), TGFβ treatment alone resulted in a doubling in the cell number (Figure 2A). Pretreatment of cells with 0.02 mg/ml MMC, on the other hand, resulted in a loss of cells averaging only 77%±16% of the original cell numbers 7 days after treatment. Pretreatment with 0.002 mg/ml MMC resulted in only a modest increase in the number of cells over 7 days that remained significantly below TGFβ alone treated cells (p<0.001). When treatment of cells with TGFβ was delayed 7 days to allow for DNA repair, MMC treated keratocytes continued to show a dose dependent decrease in the ability to replicate and express αSMA in response to TGFβ (data not shown) suggesting prolonged effects of MMC that are not repaired in culture.

To assess the effect of MMC on gene expression by quiescent keratocytes, the expression of MCP-1 and αSMA were evaluated by RT–PCR. MCP-1, is a small inducible cytokine belonging to the CC chemokine family that is involved in recruiting macrophages, memory T-cells and dendritic cells to sites of injury and is thought to play an important role in the development of fibrosis [30,31]. As shown in Figure 2B, TGFβ treated quiescent keratocytes showed increased expression of mRNA for MCP-1 and αSMA. Treatment with increasing concentrations of MMC showed a dose dependent loss of TGFβ-induced MCP-1. Interestingly, expression of TGFβ-induced αSMA mRNA showed increasing expression with higher concentrations of MMC. Overall, these findings are consistent with MMC induced DNA damage leading to abnormal cell replication and gene expression patterns.

MMC induced DNA damage and repair in culture

Since the effects of MMC on quiescent keratocytes were not reversed by extended cell culture, we next evaluated the MMC induced DNA damage and repair. As a bifunctional ankylating agent, MMC induces DNA interstand crosslinks (ICLs) that lead to phosphorylation of histone, H2AX, which recruits nuclear excision repair endonucleases that detach ICLs and repair DNA through a homologous recombination [16-18]. Antibodies specific for γH2AX, the phosphorylated form of H2AX used as a molecular marker for DNA ICLs [16], stained keratocyte nuclei that had been treated with MMC (Figure 3A). Quantification of the number of cells stained by γH2AX following MMC treatment showed a dose dependent increase with concentration of 0.07 mg/ml showing greater than 80% γH2AX staining that peaked 2 days after treatment and decreased to 40% by day 4 (data not shown). When MMC treated keratocytes were stained for Ki67, a marker of cell cycle entry and DNA repair, no Ki67 staining was detected (Figure 3B), suggesting that keratocytes did not go through a normal replication coupled DNA repair pathway. This is different from corneal fibroblasts that showed both γH2AX staining and Ki67 labeling following treatment with MMC (Figure 3C,3D, respectively).

To determine whether MMC treated keratocytes undergo DNA repair, a Comet assay was performed on MMC treated quiescent keratocytes and fibroblast cultures to detect interstrand breaks in nuclear DNA, necessary to uncouple and remove ICLs in preparation for DNA repair. In a Comet assay, DNA migrates from the nuclei, or head of the Comet, and forms a progressively longer tail depending on the number of breaks (Figure 3E, insert). Replicating cells, such as corneal fibroblasts, show a dose dependent increase in the Tail Moment of the Comet when treated with MMC and evaluated 24 h after treatment, indicating uncoupling of ICLs. By comparison, keratocytes showed a marked decrease in Tail moment 24 h after treatment with increasing doses of MMC, suggesting no DNA excision repair following MMC treatment. Overall, these data indicate that MMC dose dependently leads to DNA damage of quiescent corneal keratocytes in culture that does not undergo normal replicative DNA repair.

MMC induced DNA damage in the cornea

To determine whether MMC induced DNA damage in the cornea, rabbits were treated with MMC following scrape injury and then evaluated for the expression of γH2AX 3 h after treatment (Figure 4). Sections stained with antibodies to γH2AX (green) and counter stained with nuclear dye (red) showed staining of nuclei both in the anterior stroma (Figure 4A, arrow), posterior stroma (Figure 4B), and corneal endothelium (Figure 4B, arrow). In the very anterior stroma, keratocyte nuclei show little or no staining, most likely related to the known keratocyte apoptosis and cell death following scrape injury (Figure 4A, arrowhead). When the intensity of γH2AX staining over the nuclei was measured, very high intensity was detected over stromal keratocytes when exposed to 0.2 mg/ml MMC for 60 s (Figure 4C). There was a significant decrease in intensity (p<0.05) when the exposure was reduced from 60 s to 15 s, but the intensity of staining remained significantly greater than that detected for corneas treated with 0.02 mg/ml MMC for either 60 s or 15 s, which was not significantly different from background. Regarding differences in staining between anterior and posterior keratocytes, no significant differences were detected for both 60 s and 15 s exposures to 0.2 mg/ml MMC, suggesting all cells in the corneal stroma are equally damaged by MMC treatment in the rabbit at a given dose and exposure. It should also be noted that expression of Ki67 was not detected in corneal sections, consistent with previous studies that have evaluated later time points (data not shown) [15]. Overall, these data indicate that 0.2 mg/ml MMC (either 60 s or 15 s exposure times) is the lowest dose that induces significant DNA damage.

Effect of delayed injury on MMC treated corneas

To access repair of MMC DNA damage to quiescent keratocytes in live rabbits, eyes were treated with 0.2 mg/ml MMC for 60 s following epithelial scrape injury. Rabbits were then allowed to recover for 2 months before 100 μm deep LK injury followed by evaluation using in vivo confocal microscopy. As shown in previous studies [26,27], LK injury in control, vehicle treated eyes resulted in significant corneal scarring that was detected by in vivo confocal microscopy as intense light scattering (Scar) underlying the corneal epithelium (Epi) in XZ projections (Figure 5A). In regions of the scar, single planes (XY) showed a high density of scar fibroblasts embedded in highly light scattering extracellular matrix (Figure 5B). By contrast, eyes treated 2 months previously with 0.2 mg/ml MMC showed no corneal scaring in XZ projections (Figure 5C) and in single planes (XY) underlying the corneal epithelium showed a sparse population of keratocytes embedded in low light scattering, transparent extracellular matrix (Figure 5D). Measurement of the light scattering from the corneas also showed significantly reduced (p<0.001) corneal haze in eyes pretreated with 0.2 mg/ml MMC (Figure 5E).

The absence of corneal scarring and reduced keratocyte cell numbers was also confirmed by ex vivo confocal and nonlinear optical imaging of collagen using SHG imaging (Figure 6). In vehicle treated eyes, irregular collagen organization was detected by SHG (Figure 6A) that measured on average 84.2±27 μm in thickness. By comparison, eyes previously treated with 0.2 mg/ml MMC showed no irregular collagen deposition and only the presence of normal, broad collagen lamellae at the interface between the corneal epithelium and the stroma (Figure 6B). The nuclear density of the corneal scar in the vehicle treated eyes was also significantly greater (p<0.05) averaging 382±114 cells/mm² immediately underlying the corneal epithelium compared to the MMC treated eyes, which averaged 126±67 cells/mm². Overall, this data suggests that MMC damage to quiescent corneal keratocytes goes unrepaired in rabbits for at least 2 months after treatment.