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Published in final edited form as: Exp Eye Res. 2011 Sep 29;93(6):810–817. doi: 10.1016/j.exer.2011.09.012

Effect of TGFβ and PDGF-B blockade on corneal myofibroblast development in mice

Singh V a, MR Santhiago a, FL Barbosa a, Agrawal V a, N Singh b, BK Ambati b, SE Wilson a
PMCID: PMC3225643  NIHMSID: NIHMS330431  PMID: 21978952

Abstract

The purpose of this study was to investigate the role of transforming growth factor beta (TGFβ) and/or platelet-derived growth factor-B (PDGF-B) blockade on the differentiation of vimentin and alpha-smooth muscle actin (αSMA)-expressing myofibroblasts associated with haze in mice. Mouse corneas had haze-generating irregular PTK (phototherapeutic keratectomy) and topical treatment with the vectors. Six study groups of PTK treated corneas, with four corneas per group in each experiment, were Group 1) treated with TGFβ-KDEL vector interfering with TGFβ signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 2) treated with PDGF-B-KDEL vector interfering with PDGF signaling through anomalous sorting of cytokine bound to the expressed altered receptor; Group 3) treated with both TGFβ-KDEL vector and PDGF-B-KDEL vector to interfere with signaling of both cytokines; Group 4) empty pGFPC1 vector; Group 5) empty pCMV vector; and Group 6) no vector treatment control. At one month after surgery, the corneas were analyzed by immunocytochemistry (IHC) for central stromal cells expressing myofibroblast markers vimentin and αSMA. The stroma of corneas treated with the TGFβ-KDEL vector alone (p <0.05) or both the TGFβ-KDEL and PDGF-B-KDEL vectors (P < 0.05) had significantly lower density of vimentin-positive cells compared to the corresponding control group. The central stroma of corneas treated with the TGFβ-KDEL vector (p <0.05) or the PDGF-B-KDEL vector (p < 0.05) had lower density of αSMA-positive cells compared to the corresponding control group. The density of αSMA-positive stromal cells was also significantly lower (p < 0.05) when both the TGFβ-KDEL and PDGF-B-KDEL and vectors were applied together compared to the corresponding control groups. This study provides in situ evidence that TGFβ and PDGF-B have important roles in modulating myofibroblast generation in the mouse cornea after haze-associated injury.

Keywords: Cornea, myofibroblasts, vimentin, alpha-smooth muscle actin, corneal opacity, transforming growth factor beta, platelet-derived growth factor, corneal wound healing, gene delivery

1. Introduction

Myofibroblast generation and persistence, along with extracellular matrix deposition by these cells, is associated with the development of stromal opacity (also referred to as haze) during corneal wound healing (Lee et al., 2007; Funderburgh et al., 2003; Jester et al., 1999; Mohan et al., 2003). Myofibroblasts are fibroblastic cells that can be derived from a variety of cell precursors depending on the tissue and inciting event (Saika et al., 2010; Novo et al., 2009; Barbosa et al., 2010).

The identity of the progenitor cell(s) for myofibroblasts in the corneal stroma remains a subject of active investigation. Corneal fibroblasts, bone marrow-derived cells, or possibly even corneal epithelial cells, may give rise to corneal myofibroblasts, depending on the inciting injury, the genetic makeup of the individual, or other unknown factors (Direkze et al., 2003; Mohan et al., 2008; Barbosa et al., 2010). In vitro studies have shown that TGFβ signaling has a critical role in the differentiation of myofibroblasts from corneal fibroblasts and the persistence of myofibroblasts in culture (Tandon et al., 2010; Imanishi et al., 2000). Other studies suggested that TGFβ induces keratocyte or corneal fibroblast proliferation and myofibroblast differentiation through activation of a PDGF autocrine loop (Jester et al., 1999; Shephard et al., 2004; Zhang et al., 1999). Gene transfer studies in which PDGF-B effects were blocked in the stroma confirmed a role for PDGF in myofibroblast generation in rabbits (Kaur et al., 2009). That study suggested that PDGF-B acted at the specific point of development from vimentin+ α-smooth muscle actin− desmin− (V+A−D−) myofibroblast to V+A+D− myofibroblast. However, TGFβ blockade with the plasmid vectors was not effective in that rabbit study, likely due to genetic sequence differences between rabbit and human sequences used in designing the vectors (Kaur et al., 2009). The TGFβ receptor vector used in the prior study was designed from sequences of the human TGFβ receptor II domains 2 and 3 (GenBank: AC096921.2, Kaur et al., 2009). These human sequences are 93% concordant with mouse TGFβ II and were shown to bind mouse TGFβ in preliminary studies (unpublished data, B. Ambati, 2008). Both cytokine blockade vectors contain the KDEL endoplasmic reticulum retention signal that triggers anomalous intracellular sorting of the cytokines once they are bound to the receptor domains and, therefore, interfere with proper signaling by the cytokines once they are bound to the receptor (Kaur et al., 2009).

In the present study, we investigated the role of TGFβ and/or PDGF-B blockade using a mouse model in which myofibroblasts were identified through their expression of vimentin and α-smooth muscle actin markers.

2. Materials and methods

2.1. Animals and surgery

All animals were treated in accordance with the tenets of the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the Animal Control Committee at the Cleveland Clinic approved these studies.

Anesthesia was obtained with intraperitoneal injection of 130 μg ketamine and 8.8 μg xylazine per gram of body weight. One drop of 1% proparacaine HCl (Alcon, Ft. Worth, TX, USA) was applied topically to the eye prior to phototherapeutic keratectomy. Four C57Bl/6 female 8 to 12 week old mice were included in each group. Haze was generated in one eye by performing irregular PTK over a metallic mesh with the VISX S4 IR laser (AMO, Irvine, CA) using a previously reported method (Mohan et al., 2008).

2.2. Vectors and Animal treatment groups

Preliminary in vitro studies were performed to demonstrate efficacy of the vectors in mouse corneal fibroblasts, similar to those previously published for the pCMV.PDGFRB.23KDEL vector in rabbit corneal fibroblasts (Kaur, et al., 2009). Briefly, each vector (pGFP.TGFRBKDEL or pCMV.PDGFRB.23KDEL), or empty control vector, was transfected into primary mouse corneal fibroblasts in vitro. Immunocytochemistry demonstrated accumulation of the corresponding growth factor receptor in membrane bound organelles in the cytoplasm of the corneal fibroblasts, whereas no accumulation of receptor was noted when the corneal fibroblasts were transfected with empty control vectors (not shown). Subsequently, we also demonstrated that transfection with pCMV.PDGFRB.23KDEL, but not empty control vector, inhibited the proliferative response of first passage mouse corneal fibroblasts to 5 ng/ml mouse PDGF-AA (eBioscience, Cat. 14-8989-62, San Diego, CA) and transfection with pGFP.TGFRBKDEL, but not empty control vector, inhibited transformation of mouse corneal fibroblasts to αSMA+ myofibroblasts in response to 5 ng/ml human TGFβ-R&D Systems, Cat. 100-B-001, Minneapolis, MN)(data not shown).

Animals were divided into six treatment groups, with four animals in each group in each experiment. Each experiment was repeated and yielded consistent results. In each group, the treated eye received 30 μl of one or both vectors at a concentration of 1000 ng/μl applied to the exposed stroma immediately after PTK and every 24 hours for 4 days. At this point, the epithelium was healed in 100% of mouse eyes. The design of the vectors used to block TGFβ or PDGF signaling were previously described (Kaur et al., 2009). The study groups were 1) control group with no treatment; 2) empty pGFPC1 vector (a variant of pCMV vector); 3) empty pCMV vector; 4) both PDGF-B-KDEL (pCMV.PDGFRB.23KDEL) PDGF-B blocking vector and TGFβ-KDEL (pGFP.TGFRBKDEL) blocking vector; 5) TGFβ-KDEL vector alone; and 6) PDGF-B-KDEL vector alone. All treated eyes received one drop of 0.3% ciprofloxacin antibiotic 30 minutes after the study vectors twice a day until the epithelium was healed (Fig. 1).

Fig.1.

Fig.1

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Experimental design of the study.

2.3. Quantification of corneal haze

The level of opacity (haze) in the cornea was graded with a slit lamp (Topcon, Oakland, NJ) at one month after PTK (Fantes et al., 1990).

2.4. Tissue fixation and sectioning

Mice were euthanized at one month and the treated eye was enucleated and embedded in liquid OCT compound (Sakura FineTek, Torrance, CA) inside a tissue mold (Fisher, Pittsburgh, PA). The tissue specimens were centered so that the block was bisected and transverse sections cut from the center of the cornea through the mouse eye. Frozen tissue blocks were stored at −80°C until sectioning.

One month was selected for the later time point because this was the approximate interval after irregular PTK that haze in mice and αSMA+ myofibroblasts peaked in the mouse model (Mohan, et al., 2008).

Central corneal sections (7-micrometer thick) of the eyes were cut with a cryostat (HM 505M, Micron GmbH, Walldorf, DE, Germany). Sections were placed on 25 × 75 × 1 mm microscope slides (Superfrost Plus, Fisher) and frozen at −80°C until staining was performed.

2.5. Immunohistochemistry

Immunofluorescence staining was performed on experimental and control tissue sections as previously described (Barbosa et al., 2010). Briefly, sections from the central cornea were stained for vimentin (# 7557, Santa Cruz Biotech, Santa Cruz, CA) diluted 1:100 in phosphate buffered saline with 1% bovine serum albumin (Sigma, St. Louis, MO) or for αSMA (ab5694, Abcam, San Francisco, CA) diluted 1:50 in 1% BSA for 90 minutes. The concentration of primary antibody for vimentin was selected because at this concentration, vimentin is detected in some, but not all, keratocytes in the unwounded cornea, as we previously noted in rabbit corneas (Chaurasia, et al., 2009). Sections were then incubated with secondary antibodies (A11055 for vimentin and A11037 for αSMA, Invitrogen, Carlsbad, CA) for 60 minutes before washing with PBS three times. Immunocytochemical controls were performed by both omitting primary antibody and substituting mouse non-specific IgG1 for the primary antibody, and yielded identical results. Coverslips were mounted with Vectashield containing 4′,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories Inc., Burlingame, CA) to allow visualization of all nuclei in the tissue sections. The sections were viewed and photographed with a Leica DM5000 microscope equipped with Q-Imaging Retiga 4000RV (Surrey, BC, Canada) camera and ImagePro software. All IHC was performed at least three times to insure the results were consistent.

2.6. Quantification of cells

αSMA+ or Vimentin+ cells were counted real time at the microscope. In each case, counts of αSMA+ or vimentin+ cells were performed in central cornea and counting the number of cells per 400X full thickness column in the central cornea and averaging the counts for three adjacent fields, as previously described (Barbosa et al., 2009).

2.7. Statistical analysis

All experiments were repeated twice and the results were similar in the different runs of the experiments. Statistical analyses were performed in consultation with the Department of Quantitative Health Sciences at the Cleveland Clinic. Briefly, data were analyzed using means and standard errors, and graphical summaries were evaluated within animal groups. Analyses were performed using SAS software (version 9.1; Cary, NC). An overall significance level of 0.05 was assumed for all comparisons, and a Bonferroni (corrected significance level of 0.05/15 =0.003 was assumed for paired comparisons performed between groups if the overall p-value was statistically significant.

3. Result

3.1. Effect of cytokine blockade on vimentin expressing cells

One month after irregular PTK, the central stroma had significantly fewer vimentin-positive cells after treatment with pGFP.TGFβ-KDEL vector alone or both the PDGF-B-KDEL and TGFβ-KDEL vectors, as compared to the corresponding control groups (Fig. 2). The PDGF-B-KDEL vector alone did not have an effect on vimentin-positive stromal cells (not shown). A graph showing means and standard error of the means for vimentin is shown in Fig. 3.

Fig.2.

Fig.2

Fig.2

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Representative vimentin immunocytochemistry images of the central corneas of mice at one month after irregular PTK and treatment with cytokine blocking vectors or control vectors. The left lane shows the nuclei of all cells stained blue with DAPI. The middle lane shows vimentin-expressing cells stained green. The right lane is an overlay of the DAPI and vimentin staining. The arrows indicate some representative vimentin+ cells. A is a cornea that had no PTK or vector treatment. B is a cornea that had irregular PTK but no vector treatment. C is a control cornea that had PTK and was treated with empty pCMV vector. D is a control cornea that had PTK and was treated with empty pGFPC1 vector. E is a cornea that had PTK and was treated with the TGFβ blocking vector. F is a cornea that had PTK and was treated with both the TGFβ and PDGF-B blocking vectors. There was no effect on vimentin when corneas were treated with the PDGF-B blocking vector alone (not shown). Magnification 200X.

Fig.3.

Fig.3

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Results for the vimentin marker using a repeated measure ANOVA (mean ± SE). *Indicates the result is significant compared to the corresponding control vector(s) at p ≤0.05. The pGFP.TGFRBKDEL group had significantly fewer mean positive cells than the other groups (p<0.05), except the group containing both the pGFP.TGFRBKDEL and pCMV.PDGFRB.23KDEL vectors. The pCMV.PDGFRB.23KDEL alone group was not significantly different from the corresponding empty vector control group. Note that quantitation was performed real time at the microscope and not from captured images.

3.2. Effect of cytokine blockade on αSMA expressing cells

One month after irregular PTK, the central stroma had fewer αSMA-positive cells when treated with the TGFβ blocking vector TGFβ-KDEL or the PDGF blocking vector PDGF-B-KDEL (Fig. 4) compared with the corresponding empty vector group or the untreated control group. When both the PDGF-B-KDEL and TGFβ-KDEL vectors were applied together, the number of αSMA-positive stromal cells was also significantly lower compared to the control groups, but not significantly different from either the pCMV.PDGFRB.23KDEL alone group or the pGFP.TGFRBKDEL alone group (Fig. 5).

Fig.4.

Fig.4

Fig.4

Fig.4

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Representative α-smooth muscle actin (αSMA) immunocytochemistry images of the central corneas of mice at one month after irregular PTK and treatment with cytokine blocking vectors or control vectors. Left lane shows the nuclei of all cells stained blue with DAPI. The middle lane shows αSMA-expressing cells stained red. The right lane is an overlay of the DAPI and αSMA staining. The arrows indicate some representative αSMA+ cells. A is a cornea that had no PTK or vector treatment. B is a cornea that had irregular PTK but no vector treatment. C is a control cornea that had PTK and was treated with empty pCMV vector. D is a control cornea that had PTK and was treated with empty pGFPC1 vector. E is a cornea that had PTK and was treated with the TGFβ blocking vector. F is a cornea that had PTK and was treated with the PDGF-B blocking vector. G is a cornea that had PTK and was treated with both the TGFβ and PDGF-B blocking vectors. Magnification 200X

Fig.5.

Fig.5

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Results for the αSMA marker using a repeated measure ANOVA (mean ± SE). *Indicates the result is significant compared to the corresponding control vector(s) at p ≤0.05. “Control” is a cornea from the group that had irregular PTK without vector treatment. Treatment with either the pGFP.TGFRBKDEL vector or the pCMV.PDGFRB.23KDEL, or with both the pGFP.TGFRBKDEL vector and the pCMV.PDGFRB.23KDEL vector, significantly decreased αSMA+ cells compared to the corresponding empty vector controls (p<0.05). Note that quantitation was performed real time at the microscope and not from captured images.

3.3. Evaluation of haze at one month after irregular PTK

At the slit lamp, stromal haze was noticeably less in groups treated with the TGFβ-KDEL vector or PDGF-B-KEL vector alone, or both vectors together, compared to either vector control group or the PTK group that received no vectors.

These experiments were performed twice and the results were similar in the two experiments. The results of one of these experiments are provided for vimentin and α-smooth muscle actin.

4. Discussion

Many studies have shown that α-smooth muscle actin (αSMA)-positive myofibroblasts are associated with corneal opacity (referred to clinically as haze) associated with injuries, surgeries and infection (Jester, et al., 1999; Kim et al., 1999; Funderburgh, et al., 2003; Mohan, et al., 2003; Netto et al., 2006a, 2006b). After lacerations or incisions, myofibroblast cells function to contract the stromal wound (Jester, et al., 1999; Desmouliere et al., 1995). The function of myofibroblasts broadly populating the subepithelial stroma after surface ablation procedures is less clear, and may merely be a function of a response to surgery-induced structural and functional defects of the epithelial basement membrane that result in increased TGFβ penetration into the anterior stroma from the overlying epithelium (Netto et al, 2006a).

Cytokines and growth factors play important roles in the development and persistence of myofibroblasts in the cornea. TGFβ and PDGF have been shown to have critical functions in modulating the differentiation of αSMA-positive myofibroblast cells from precursor cells (Jester et al., 1997, 1999a, 1999b; Zhang et al., 1999; Shephard et al., 2004; Barbosa et al., 2010). TGFβ and PDGF are produced at high levels by corneal epithelial cells and TGFβ is also produced by stromal cells, albeit at lower levels (Wilson, et al., 1994; Kim, et al., 1999). TGFβ can regulate the generation of myofibroblasts from either keratocyte-derived corneal fibroblasts (Mazur, et al., 1996; Jester, et al., 1999a) or bone marrow-derived cells (V. Singh and S.E. Wilson, unpublished data, 2011).

The present study used plasmid vectors that express genetically altered receptors that bind TGFβ or PDGF-B, and include a KDEL endoplasmic reticulum retention signal (Kaur et al., 2009) that interferes with normal signaling of the bound growth factors. The TGFβ receptor vector was designed from the human TGFβ receptor II domains 2 and 3 sequences. The PDGF vector expressed a subunit of PDGF receptor b (domains 2–3). These vectors were previously used in a study in rabbit corneas, but only the PDGF B-blocking vector was effective in that species due to sequence differences in the rabbit (Kaur et al., 2009).

This study demonstrates that stromal blockade of TGFβ signaling decreases the density of vimentin-positive (vimentin+) cells in the stroma after opacity-generating corneal injury. Keratocytes in the unwounded cornea express basal levels of vimentin (Kivela and Uusitalo, 1998; Mimura et al., 2008), but the number of vimentin+ stromal cells, and the level of expression of vimentin in many stromal cells, increases after opacity-generating corneal injury (Chaurasia, et al., 2009). Importantly, the concentration of primary antibody used for immunocytochemical detection of vimentin in this study was selected in preliminary experiments to be less than that required to detect vimentin expression in most keratocytes in unwounded corneas (Fig. 2A), but to allow detection in cells such as myofibroblasts where the expression of vimentin is higher after irregular PTK (Fig. 2B) or other haze-generating injuries (Chaurasia, et al., 2009). Vimentin+ cells can serve as keratocyte-derived precursors in the formation of vimentin+ αSMA+ myofibroblasts (V+A+ myofibroblasts) in the cornea (Chaurasia, et al., 2009). Blockade of PDGF-B function alone has no effect on the formation of V+ stromal cells. However, TGFβ blockade or PDGF-B blockade decreases the generation of αSMA+ myofibroblasts after opacity-generating corneal injury. Taken together, these results suggest that PDGF-B modulates the V+A− myofibroblast precursor to V+A+ myofibroblast transition during myofibroblast development (Chaurasia, et al., 2009), as was previously suggested by studies in the rabbit model (Kaur et al., 2009). However, TGFβ modulates both the increase in strongly vimentin+ stromal cells and the V+A− myofibroblast precursor to V+A+ myofibroblast transition after opacity-generating corneal injury. Experiments are in progress to determine whether PDGF-B and TGFβ have similar roles in regulating myofibroblast development from bone marrow-derived precursor cells (Singh V and Wilson SE, unpublished data, 2012).

Importantly, many stromal cell types, and even epithelial cells remaining beyond the wound edge or regenerating during the early days after surgery, could be transfected by the vectors used in this study. Thus, we are uncertain which cells the vectors used in this study are modulating. The author’s hypothesize this includes residual peripheral and posterior keratocytes that have not undergone apoptosis (Wilson, et al., 1995) and their progeny corneal fibroblasts, some of which are likely precursors to corneal myofibroblasts (Masur, et al., 1996; Jester, et al., 1999a). However, since the vectors were applied for four days after surgery, bone marrow-derived cells invading the cornea were also likely transfected with the vectors. Studies have shown that some bone marrow-derived cells can also be precursors to myofibroblasts in the cornea (Barbosa, et al., 2010). Whether or not blockade of TGFβ or PDGF function alters differentiation of corneal myofibroblasts that originate from bone marrow-derived cells is currently under investigation in our laboratory in chimeric mice in which bone marrow-derived cells express green fluorescent protein.

The haze response in mice, even with the irregular PTK method used in these studies (Mohan, et al., 2008), is less than it is in rabbits after comparable PTK or photorefractive keratectomy to correct nine diopters of myopia. We did, however, qualitatively note decreased haze in corneas that had irregular PTK and were treated with either the TGFβ- or PDGF-B-blocking vectors, or the two blocking vectors together, compared to the control groups that had irregular PTK and treatment with empty vectors or treatment with no vectors. At time points earlier than one month, when many vimentin+ cells (including immature myofibroblasts), but few αSMA+ myofibroblasts are detected in the stroma, very little haze is detected at the slit lamp in the cornea. Haze becomes easily detectible when many αSMA+ myofibroblasts are present after irregular PTK. This is similar to our findings in rabbits (Chaurasia, et al. 2009) and suggests that vimentin+αSMA-myofibroblasts precursors do not contribute significantly to optical haze, but do contribute once they further differentiate into vimentin+αSMA+ mature myofibroblasts.

Studies to define the signaling pathways that regulate differentiation of corneal myofibroblasts are likely to have important clinical implications. Depending on the situation, it is often desirable to inhibit or augment the development of opacity-associated myofibroblasts in the cornea. For example, it would be advantageous to circumferentially and uniformly increase myofibroblast density at the donor-host interface after penetrating keratoplasty to increase wound strength. Conversely, it is desirable to decrease opacity-associated myofibroblast generation after photorefractive keratectomy or phototherapeutic keratectomy, or after microbial infections. At the present time, mitomycin C, a non-specific blocker of corneal cell proliferation that also decreases keratocyte density when it inhibits myofibroblast generation (Netto, et al., 2006b), is the only medication used clinically to modulate myofibroblasts. It can be anticipated that a fuller understanding of cytokine regulation of myofibroblast generation and death will lead to more selective and safer pharmacologic agents to increase or decrease myofibroblast density in the cornea.

Highlights.

  • TGFβ and PDGF-B modulate the development of corneal myofibroblasts in vivo

  • TGFβ increases vimentin+α-smooth muscle actin- (V+A−) myofibroblast precursors

  • PDGF-B modulates the transition of V+A− precursors to V+A+ myofibroblasts

Acknowledgments

Supported in part by US Public Health Service grants EY10056 and EY015638 from the National Eye Institute, National Institutes of Health, Bethesda, MD and Research to Prevent Blindness, New York, NY.

Footnotes

Proprietary interest statement: None of the authors have any proprietary or financial interests in the topics discussed in this manuscript

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