Overexpression of CDKN1B Inhibits Fibroblast Proliferation in a Rabbit Model of Experimental Glaucoma Filtration Surgery

Abstract

Purpose.: To investigate the potential antiproliferative effect of cyclin-dependent kinase inhibitor 1B (CDKN1B) overexpression in a rabbit model of glaucoma filtration surgery (GFS).

Methods.: The recombinant adenovector expressing exogenous CDKN1B was delivered to Tenon's capsule by subconjunctival injection during unilateral filtration surgery. The time course of CDKN1B expression was monitored by immunohistochemistry and Western blot analysis. Evaluation of proliferating activity was performed by proliferating cell nuclear antigen (PCNA), argyrophilic nucleolar organizing region (AgNOR) staining, and fibroblast-specific protein 1 (FSP-1). Cyclin-dependent kinase 2 (Cdk2) and Cdk4 expression were detected with immunohistochemical analysis.

Results.: The overexpression of CDKN1B in Tenon's capsule was monitored throughout the experimental period. Immunoreactivity to CDKN1B was mainly observed in the nucleus of fibroblasts. The increased expression of CDKN1B in sclera was detected up to 21 days after viral infection, whereas the level of CDKN1B protein in corneal stroma was not significantly increased. The overexpression of CDKN1B induced a significant decrease in AgNOR number/nucleus and area/nucleus, PCNA staining, FSP-1 positive cells, and the decreased expressions of Cdk2 and Cdk4, as evidenced by nuclear and cytoplasmic immunoreactivity to Cdk2 and Cdk4 antibodies in positive fibroblasts.

Conclusions.: The persistent overexpression of CDKN1B mediated by the recombinant adenovector expressing exogenous CDKN1B in Tenon's fibroblasts after GFS may lead to the inhibition of fibroblast proliferation and the downregulation of Cdk2 and Cdk4 activity, thereby reducing the severity of scar formation and the surgical outcome.

Introduction

Glaucoma filtration surgery (GFS) is the most widely used procedure for maintaining a suitable target intraocular pressure (IOP) for glaucoma patients. However, excessive scarring of the conjunctiva at the bleb and sclerostomy sites after GFS often leads to the failure of the surgery. ^1–4 It has been proved that the proliferation of fibroblasts from the subconjunctival space is the major cause of the scarring process. ^5,6  

Both positive and negative regulators of cell-cycle activity are the critical regulators of cell proliferation and differentiation. The positive regulators include cyclins and cyclin-dependent kinases (Cdks), whereas the negative regulators consisted in cyclin-dependent kinase inhibitors (CKIs). ^7–9 Cyclin-dependent kinase inhibitor 1B (CDKN1B, also known as p27^Kip1 or p27) is a well-characterized CKI that belongs to the Cip/Kip family, which is an important negative modulator of cell-cycle progression. The protein level of CDKN1B is highest in quiescent cells and declines as cells are stimulated to reenter the cell cycle. ^10,11 Once activated, CDKN1B protein regulates cell-cycle progression that specifically inhibits cyclin E/Cdk2, cyclin A/Cdk2, and cyclin D/Cdk4/6 complexes, which are necessary for DNA replication, and thereby abrogates their catalytic activity, leading to potent arrest at the G₁/S-phase transition. ^12–14 In S-phase, CDKN1B is phosphorylated at Thr-187 by cyclin E/Cdk2 and then ubiquitylated in proliferating cells by SCF^Skp2 (S-phase kinase-associated protein 2), relieving the blockade of cell-cycle progression. ^15,16  

CDKN1B, as one of the most widely distributed CKIs in most human tissues, is expressed both in proliferating and differentiated cells. In our previous study, ¹⁷ we performed a rabbit model of experimental GFS that locally applied adenovirus-mediated CDKN1B (Ad-CDKN1B). Ad-CDKN1B could induce an ocular hypotensive response and increase bleb survival during a 28-day period. However, Ad-CDKN1B did not result in a variety of toxicities induced by mitomycin C, including wound leakage, corneal erosion, and chronic hypotony. The blebs displayed histologic features of marked reduction in total cellularity, consisted of the decreased number of conjunctival epithelial cells and goblet cells, and the noted reduction in subconjunctival scar tissue following Ad-CDKN1B delivery. Ultrastructurally, Tenon's fibroblasts showed the reduced activation and increased apoptosis at the surgical sites. We speculate that the prolonged wound healing is associated with the inhibition of subconjunctival fibroblast proliferation. Recent studies have suggested a physiologic role of CDKN1B as a regulator of fibroblast growth in the pathogenesis of proliferative diseases. In vitro experiments have shown that overexpression of CDKN1B efficiently blocked fibroblast activity in cultured Tenon's fibroblasts and limited fibroblast proliferation. ¹⁸ Reduced Cdk2 activity and the decline in cell proliferation that took place at late time points after angioplasty correlated with a marked induction of CDKN1B. ^19–21 The nuclear accumulation of CKI was associated with a quiescent and static phenotype. ^22,23  

Thus, we hypothesize that CDKN1B plays an important role in modulating Tenon's fibroblast proliferation after GFS. In the present study, we used a rabbit model of GFS to analyze the ectopic expression of CDKN1B and attempted to elucidate the potential mechanism of antiproliferative effects from CDKN1B. 

Methods

Construction of Recombinant Adenovirus Vector

Construction of the recombinant adenovirus vectors has been described in detail elsewhere. ^17,24 Essentially, full-length human CDKN1B cDNA was cloned into a shuttle vector containing the human cytomegalovirus immediate-early promoter and a plasmid that contains replication adenovirus genome. Adenoviruses were grown and propagated in the HEK-293 cell line. The presence of CDKN1B cDNA in the viral genome was further purified using standard protocols. Viral particle number of purified recombinant adenoviral vectors was determined using HPLC quantification and reported as plaque-forming units (pfu), which was 2.1 × 10¹² pfu/mL. 

CDKN1B Delivery for GFS

A randomized, controlled, masked-observer study was conducted in a rabbit model of GFS. Adult albino rabbits weighing between 2 and 3 kg were anesthetized with an intramuscular injection of ketamine (35–45 mg/kg) and xylazine (5–10 mg/kg). Surgical eyes underwent a unilateral sclerectomy using a limbal-based triangle scleral flap with a peripheral iridectomy approach, the details of which are described elsewhere. ¹⁷  

A total of 96 rabbits were randomly divided into Ad-CDKN1B and control groups: 48 were used for Western blot analysis (six in each group) and 48 for morphometric analysis (six in each group). The Ad-CDKN1B group was delivered as 8.0 × 10¹¹ pfu/mL (100 μL volume) Ad-CDKN1B into Tenon's capsule by a subconjunctival injection at the superolateral quadrant during filtration surgery. Injection of PBS served as a control. After surgery, 1% atropine sulfate ophthalmic ointment, neomycin sulfate, and dexamethasone were applied to the eye. 

All procedures in these animal experiments were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research, and approved by the Committee for Animal Research, Xi'an Medical University. 

Argyrophilic Nucleolar Organizing Region Staining

Argyrophilic nucleolar organizing region (AgNOR) histochemical staining was performed using a previously described modified silver-staining technique. ²⁵ In brief, the eyes were enucleated together with the conjunctiva to preserve the bleb on days 7, 14, 21, and 28 after surgery. Formalin-fixed, paraffin-embedded tissue sections were cut at 5 μm, deparaffinized in xylene, rehydrated in graded ethanol, and rinsed in distilled deionized water. Slides were incubated for 25 to 30 minutes at room temperature in the dark with freshly made AgNOR staining solution, consisting of 0.02 g gelatin in 1 mL 1% formic acid and 1 g silver nitrate in 2 mL of distilled water. Following AgNOR staining, slides were rinsed with distilled deionized water, dehydrated with graded ethanol, cleared with xylene, and mounted in synthetic medium. 

Morphometric analysis and quantification of AgNORs were performed by microscopy at ×400 magnification with an image analysis system (KS400; Carl Zeiss Microscopy, Jena, Germany). On each slide, AgNORs were counted in 100 randomly selected nuclei of fibroblasts. Mean nucleus area, AgNOR area/cell, AgNOR number/cell, and AgNOR ratio (AgNOR area/cell divided by the nucleus area) were then determined on the basis of averaging the counts within these 100 cells as shown by previous reports. ^26,27 Slides were examined by two independent evaluators. 

Immunohistochemical Analysis

For immunohistochemical analysis, 5-μm sections were deparaffinized and treated by two rounds of microwave heating for 5 minutes in 10 μM sodium citrate (pH 6). Endogenous peroxidase was blocked by incubation with 0.3% H₂O₂. Slides were blocked in 10% normal goat serum in PBS for 30 minutes and then stained for 1 hour with an anti-human proliferating cell nuclear antigen (PCNA) monoclonal antibody, rabbit polyclonal anti–fibroblast-specific protein-1 (FSP-1)/S100A4 (Dako, Glostrup, Denmark), anti-human CDKN1B monoclonal antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA), anti-human Cdk2 monoclonal antibody, and anti-human Cdk4 monoclonal antibody (Sigma, St. Louis, MO), respectively. They were subsequently incubated with a biotinylated goat anti-mouse secondary reagent (Sigma) and with horseradish peroxidase–labeled streptavidin (Zhongshan Biotechnology, Beijing, China). Bound antibodies were visualized with 0.02% 3,3′-diaminobenzidine tetrahydrochloride. The sections were counterstained with hematoxylin. 

Positive immunostaining was defined as nuclear or cytoplasmic staining of Tenon's cells. The percentages of the positive cells in total fibroblasts were calculated by examining four random high-power fields of each slide. ²⁸ In addition, immunoreactions were scored on each tissue slide for protein expression based on a semiquantitatively scoring system that measured percentage positive fibroblasts (0, none; 1, <10%; 2, 10%–50%; 3, 50%–80%; 4, >80%) and cellular localization (cytoplasm, nucleus, or combination thereof). ²⁹  

Western Blot Analysis

Western blotting was carried out as previously described. ¹⁷ In brief, the tissues at surgical sites including Tenon's capsule, corneal stroma, and superficial scleral stroma were peeled carefully after enucleating, and the conjunctival and corneal epithelium were separated completely from Tenon's capsule and corneal stroma, respectively. The samples were collected by scraping with a spatula in 10 mL of cold medium containing 50 mM 3-(N-morpholino) propanesulfonic acid (pH 7.0), 10 mM NaF, 1 mM EDTA, 0.3 M sucrose, 0.4 mM Pefabloc, 10⁻² g/L aprotinin, 2 × 10⁻³ g/L leupeptin, and 10⁻³ g/L pepstatin A. 

After centrifugation at 2500g for 2 minutes to remove debris, an equal protein amount of cell lysates was separated on SDS-PAGE and transferred onto nitrocellulose membranes. The membranes were blocked in an ice-cold lysis bufer (10 mM Tris-Cl, 1% SDS, and 1 mM Na₂VO₄, pH 7.4). The total protein concentration was determined by the bicinchoninic acid assay method (Pierce, Rockford, IL). The blots were incubated with rabbit monoclonal antibodies to CDKN1B (Santa Cruz Biotechnology, Inc.) for 2 hours at room temperature. β-Actin (Sigma) served as an internal positive control. Reactions were visualized with a suitable secondary antibody conjugated with horseradish peroxidase using enhanced chemiluminescence reagents. 

Statistical Analysis

Statistical analysis was carried out using commercial statistical software (SPSS Statistical Program, version 13.0 for Windows; SPSS, Inc., Chicago, IL). Group differences were evaluated by one-way ANOVA or Student's t-test. The independent samples nonparametric test was used to analyze scores. A value of P < 0.05 indicates a statistically significant difference. 

Results

CDKN1B Protein Expression at the Surgical Sites

Immunohistochemstry and Western blot analysis were used to observe fibroblast proliferation at surgical sites following Ad-CDKN1B delivery. Figures 1A and 1B showed the time-course data of CDKN1B expression in Tenon's capsule by Western blot analysis. The CDKN1B level was higher obtained by Ad-CDKN1B application than that by placebo on day 7 after surgery (P = 0.001). A further increase of CDKN1B expression levels was recorded on day 14 (P = 0.000). The elevated CDKN1B protein in Ad-CDKN1B–treated eyes was still detectable after 28 days (P = 0.001). 

Figure 1. 

 

Overexpression of CDKN1B in Tenon's capsule. The elevated levels of CDKN1B protein are detected using Western blot throughout the experimental period, which the highest protein level is observed on day 21 (A, B). Immunohistochemistry analysis further confirms the overexpression of CDKN1B, which is localized in the nucleus of Tenon's fibroblasts (arrows) (Ad-CDKN1B group, C–F; control group, G–J; ×200). There are continuously high scores during the experimental period (K). Means ± SEM (n = 6). *P < 0.05, **P < 0.01. C, conjunctiva; TC, Tenon's capsule.

Immunohistochemical staining for CDKN1B in cells was scored semiquantitatively based on the number of CDKN1B-positive fibroblasts. In the present study, immunoreactivity to CDKN1B was mainly observed in the nucleus of fibroblasts. The scores were significantly elevated on day 7, in which the mean percentage of positive cells with Ad-CDKN1B was 85.08% vs. 39.53% of placebo (mean score of immunoactivity, 3.78 vs. 2.11 P = 0.000) (Figs. 1C, 1G, 1K). CDKN1B level induced by Ad-CDKN1B was further increased on day 14. The mean percentage of positive cells was 76.19% vs. 21.05% of placebo (mean score, 3.56 vs. 1.83, P = 0.000) (Figs. 1D, 1H, 1K). The score remained elevated up to day 28, in which the mean percentage of Ad-CDKN1B was 72.66% vs. 34.92% of placebo (mean score, 3.05 vs. 2.05, P = 0.000) (Figs. 1F, 1I–K). 

To better understand the roles of Ad-CDKN1B at surgical sites, we also monitored the time course of CDKN1B expression in corneal stroma and superficial scleral stroma (Fig. 2). In scleral stroma, CDKN1B overexpression following Ad-CDKN1B delivery was detected in comparison with placebo on day 7 (P = 0.016). Furthermore, the elevated protein level was maintained up to day 21 (P = 0.000). However, no difference of CDKN1B expression induced by Ad-CDKN1B and placebo was detected on day 28 (P = 0.781), although the densitometric signal remained relatively high by Western blot (Figs. 2A, 2C). The immunoreactivity to CDKN1B was mainly observed in the nucleus. As expected, the CDKN1B expression using immunohistochemistry was consistent with Western blot during the 28-day period (Figs. 2G–J). 

Figure 2. 

 

CDKN1B expression in corneal stroma and superficial scleral stroma of surgical sites. There is no increase in protein expression in corneal stroma throughout the experimental period (A, C). The immunostaining in fibroblasts is not observed either in nucleus or cytoplasm (Ad-CDKN1B, E; control, F). CDKN1B overexpression is detected in superficial scleral stroma up to day 21 (B, D). The immunostaining is localized in the nucleus of positive cells (Ad-CDKN1B, G, I; control, H, J; 200×). *P < 0.05, **P < 0.01.

In corneal stroma, CDKN1B expressions were similar by Ad-CDKN1B delivery and placebo throughout the experimental period (Figs. 2B, 2D) using both Western blot and immunohistochemstry. The immunohistochemical staining in fibroblasts was consistently almost absent in nucleus and cytoplasm (Figs. 2E, 2F). 

Argyrophilic Nucleolar Organizing Region Histologic Evaluation of Wound Healing

AgNORs are nucleolar substructures that are associated with ribosomal RNA transcription. It has been demonstrated that AgNOR count is a good marker of proliferation. In the present study, the tissues were stained with AgNOR to give an overall impression to determine proliferative fibroblasts at surgical sites. Histologic profiling revealed mild or moderate fibrotic response with extracellular matrix (ECM) deposition in Tenon's capsule following Ad-CDKN1B delivery (Figs. 3A–D). The sclerotomy site, in contrast, showed massive subconjunctival scarring, which consisted of dense ECM in placebo (Figs. 3E–H). 

Figure 3. 

 

Histochemical staining for AgNOR at surgical sites. Histologic features show less proliferation (A–D, ×100) and less AgNOR number of cells (×400) in Tenon's capsule following Ad-CDKN1B delivery in comparison with placebo (E–H). S, sclera.

The quantitative evaluation of AgNORs was carried out by morphometric analysis, by counting the number occupied by the silver-stained structures. On day 7, the mean AgNOR number/nucleus was significantly decreased following Ad-CDKN1B delivery compared with placebo (95% confidence interval [CI] for Ad-CDKN1B, 1.93–2.84; for placebo, 3.53–4.58 P = 0.000). The decreased number/nucleus was maintained throughout the experimental period, and on day 28, the difference was still significant (95% CI for Ad-CDKN1B, 1.95–3.15; for placebo, 2.98–4.16; P = 0.000) (Table). 

Table. 

 

Comparison of the Mean AgNOR Area and Number between Ad-CDKN1B and Placebo-Treated Eyes

Day	AgNOR Area, μm2			AgNOR Number			Nucleus Area, μm2			AgNOR Ratio, %
	Ad-CDKN1B	PBS		Ad-CDKN1B	PBS		Ad-CDKN1B	PBS		Ad-CDKN1B	PBS
7	0.94 ± 0.08	1.36 ± 0.11	0.004	2.39 ± 0.13	4.06 ± 0.12	0.000	15.65 ± 0.46	44.62 ± 0.86	0.000	6.16 ± 0.26	8.35 ± 0.30	0.000
14	1.03 ± 0.07	1.78 ± 0.12	0.000	2.85 ± 0.11	4.57 ± 0.11	0.000	15.51 ± 0.26	48.09 ± 0.96	0.000	5.86 ± 0.22	7.37 ± 0.41	0.003
21	1.02 ± 0.09	1.86 ± 0.08	0.000	2.65 ± 0.14	4.40 ± 0.15	0.000	17.67 ± 0.46	45.19 ± 0.65	0.000	5.80 ± 0.29	7.78 ± 0.35	0.000
28	0.79 ± 0.07	2.02 ± 0.11	0.000	2.58 ± 0.16	3.81 ± 0.12	0.000	20.08 ± 0.61	51.45 ± 1.06	0.000	6.16 ± 0.33	9.78 ± 0.32	0.000

 Means ± SEM (n = 6).

Comparison of the nuclear area of AgNOR between Ad-CDKN1B and placebo revealed that there was a significant decrease with Ad-CDKN1B delivery on day 7 (95% CI for Ad-CDKN1B, 0.77–1.10 μm²; for placebo, 1.13–1.59 μm²; P = 0.004). The decrease was maintained for 28 days (95% CI for Ad-CDKN1B, 0.64–0.93 μm²; for placebo, 1.78–2.25 μm²; P = 0.000) (Table). 

PCNA Expression of Tenon's Fibroblasts

PCNA is another marker of cell proliferation in fibroblasts that has a very long half-life. In the present study, PCNA expression relative to CDKN1B was significantly reduced compared with placebo throughout the experimental period. This reduction in nuclear immunoreactivity was representative of the inhibition on proliferation and repair mechanics. 

PCNA expression revealed a decrease of PCNA accumulation on day 7 after Ad-CDKN1B delivery (41.18% of mean PCNA-positive cells of Ad-CDKN1B vs. 83.33% of placebo; mean score, 2.17 vs. 3.50, P = 0.000) (Figs. 4A, 4E, 4I). To day 14, the decrease was still significant (52.38% of Ad-CDKN1B vs. 87.10% of placebo; mean score, 2.72 vs. 3.72, P = 0.000) (Figs. 4B, 4F, 4I), and the decreased expression was maintained up to day 28 (32.26% of Ad-CDKN1B vs. 55.36% of placebo; mean score, 2.00 vs. 2.72, P = 0.000) (Figs. 4D, 4H, 4I). 

Figure 4. 

 

Downregulation for PCNA in Tenon's capsule. The serial panels show the decreased expression for PCNA in Tenon's cells following Ad-CDKN1B delivery (A–D) in comparison with placebo (E–H, ×200). (I) Immunoactivity scores for PCNA. Means ± SEM (n = 6). *P < 0.05, **P < 0.01.

FSP-1 Expression of Tenon's Fibroblasts

FSP-1 is a member of the S100 family of cytoplasmic calcium-binding proteins, which is expressed on fibroblasts and cells that are transitioning into fibroblasts. It regulates cellular motility through a direct interaction with myosin-IIA. FSP-1 is identified by subtractive and differential mRNA hybridization as a gene expressed in fibroblasts but not in epithelial cells. ³⁰ In the present study, a few FSP-1–positive cells were found scattered at the surgical sites with Ad-CDKN1B delivery throughout the experimental period, whereas the positive cells were abundant with placebo. 

FSP-1–positive cells, staining in fibroblast cytoplasm, revealed a significant decrease on day 7 after Ad-CDKN1B delivery (38.10% of mean positive cells of Ad-CDKN1B vs. 81.82% of placebo; mean score, 2.22 vs. 3.72, P = 0.000) (Figs. 5A, 5E, 5I). To day 14, only a small fraction (29.17%) of fibroblasts was constituted by FSP-1–positive cells with Ad-CDKN1B, but the percentage of positive cells (77.55%) was still high with placebo. The mean score of immunoactivity was 1.94 of Ad-CDKN1B vs. 3.89 of placebo (P = 0.000) (Figs. 5B, 5F, 5I). The low percentage of positive cells was maintained over the experimental time course (35.71% of Ad-CDKN1B vs. 84.85% of placebo on day 28; mean score, 2.00 vs. 3.83, P = 0.000) (Figs. 5D, 5H, 5I). 

Figure 5. 

 

Expression for FSP-1 in Tenon's fibroblasts. FSP-1–positive cells are decreased in Ad-CDKN1B. The number of FSP-1–positive fibroblasts was significantly suppressed by Ad-CDKN1B (A–D) compared with placebo (arrows, E–H, ×200). (I) Immunoactivity scores for FSP-1. Means ± SEM (n = 6). *P < 0.05, **P < 0.01.

Cdk2 and Cdk4 Expression of Tenon's Capsule Fibroblasts

Cdk2 and Cdk4 expression has been largely implicated in induction of cell proliferation. Immunohistochemical analysis revealed that Cdk2 expressions relative to CDKN1B were consistently present, but at a much lower level of Cdk2 over the entire time course. Cdk2 expression was significantly decreased following Ad-DKN1B delivery on day 7, and the mean percentage of Cdk2-positive fibroblasts was 53.65% of Ad-CDKN1B vs. 78.14% of placebo (mean score, 2.67 vs. 3.44, P = 0.000). Interestingly, both nuclear and cytoplasmic immunoreactivity to Cdk2 were observed in positive cells of Ad-CDKN1B, whereas both intense cytoplasmic immunoreactivity and nuclear staining were shown in fibroblasts of placebo (Figs. 6A, 6E, 6I). Then Cdk2 expression showed a relatively small reduction on following days (Figs. 6B, 6C, 6F–J). On day 28, the significant difference remained between Ad-CDKN1B and placebo (46.82% of mean Cdk2-positive cells of Ad-CDKN1B vs. 71.68% of placebo; mean score, 2.44 vs. 3.28, P = 0.000), as evidenced by nuclear and cytoplasmic immunoreactivity to Cdk2 antibodies in positive cells between Ad-CDKN1B and placebo (Figs. 6D, 6H, 6I). 

Figure 6. 

 

Downregulation for Cdk2 in Tenon's capsule. Cdk2 expression decreases treated with Ad-CDKN1B (A–D) in comparison with placebo (arrows, E–H, ×200). (I) The immunoactivity scores for Cdk2. Means ± SEM (n = 6). *P < 0.05, **P < 0.01.

Similar to Cdk2, the expression of Cdk4 following Ad-CDKN1B delivery was significantly lower than that of placebo during the 28-day period (Fig. 7). The mean percentage of Cdk4-positive fibroblasts was 42.11% of Ad-CDKN1B and 66.67% of placebo on day 7 (mean score, 2.33 vs. 2.83, P = 0.005). Both nuclear and cytoplasmic immunoreactivity to Cdk4 were observed in positive cells in the two groups (Figs. 7A, 7E, 7I). Then Cdk4 expression was maintained at the lower level on following days (Figs. 7B–E). The mean percentage of Cdk4-positive cells was 51.72% of Ad-CDKN1B vs. 89.79% of placebo at day 28 (mean score, 2.50 vs. 3.72, P = 0.000), during which the nuclear and cytoplasmic immunostainings were still observed in Cdk4-positive cells (Figs. 7D, 7H, 7I). 

Figure 7. 

 

Cdk4 expression at surgical sites. The decreased expression for Cdk4 is observed with Ad-CDKN1B (A–D) in comparison with placebo (arrows, E–H, ×200). (I) The quantitative scores for Cdk4. Means ± SEM (n = 6). *P < 0.05, **P < 0.01.

Discussion

The filtration failure due to excessive postoperative scarring is the major challenge to the control of IOP and arrest of glaucomatous damage after GFS. The human Tenon's fibroblasts are considered to play a principal role in modulating the proliferation, migration, and synthesis of the ECM after the filtration surgery. Thus, inhibiting the proliferation of Tenon's fibroblasts during the wound healing is an important antiscarring strategy. ^1–4,17,18  

The importance of CDKN1B in regulating cell proliferation has been demonstrated in varied animal models. It has been shown that there was markedly decreased expression of CDKN1B in the corneal endothelium cells after injury in a rat model of connexin43 knockdown, ³¹ whereas the adenovirus-mediated overexpression of CDKN1B inhibited vascular smooth muscle cells proliferation, ³² and led to cell growth arrest in human renal carcinoma cells. ^33,34 Wang et al. ¹⁸ suggested that siRNA-mediated gene silencing of Skp2 induced increased CDKN1B protein levels, and subsequently inhibited the proliferation of rabbit Tenon's fibroblasts (rTFs) after GFS. Thus, it is presumed that the decreased cell proliferation was associated with elevated CDKN1B activity in conjunctival epithelial cells and rTFs, and CDKN1B may be a significant prognostic factor determining the proliferation. 

In the present study, we distinguished Tenon's fibroblasts stained with AgNOR, PCNA, Cdk2, and Cdk4 based on morphologic features and localization. The positively stained fibroblasts were further confirmed by FPS-1 staining experiments. We have identified that Ad-CDKN1B induces an increase of the CDKN1B protein level in Tenon's fibroblasts after sclerectomy in rabbits. The sustained overexpression of CDKN1B via adenovirus-mediated gene transfer leads to a persistence of inhibition of fibroblast proliferation throughout the time course after surgery. 

It has been demonstrated that two pertinent factors influence CDKN1B function: intracellular concentration and subcellular localization. ³⁵ CDKN1B activity is intimately associated with its nuclear localization in positive Tenon's fibroblasts of surgical sites, as observed in our study upon Ad-CDKN1B treatment. The nuclear localization of CDKN1B protein indicates the antiproliferative effects because the nuclear CDKN1B inhibits cyclin E-Cdk2. ³⁶ Furthermore, the nuclear export of CDKN1B has been shown to be dependent on the S10 phosphorylation of CDKN1B, and the phosphorylation regulates the reentry of quiescent cells into the cell cycle. ^35,37  

Wen et al. ³⁸ showed that adenovirus-mediated overexpression of CDKN1A (also named p21^Cip1 or p21), another Cip/Kip family member, in Tenon's fibroblasts inhibited cell proliferation, whereas no expression of CDKN1A protein was observed in scleral fibroblasts or cornea. Our data suggest that adenovirus-mediated overexpression of CDKN1B in superficial scleral stroma was observed in the most time course, although the expression level was markedly lower compared with that in Tenon's fibroblasts. These results indicate that Ad-CDKN1B may be delivered to the surrounding region of scleral flap, where it inhibits fibroblast proliferation. Yoshida et al. ³⁹ suggested that the disappearance of CDKN1B was well correlated with cell proliferation in the corneal epithelium after injury. Cdk4 and CDKN1B regulate proliferation in corneal endothelial cells by regulating the cell-cycle progression. In our in vivo study, no evidence of CDKN1B overexpression in cornea fibroblasts was found using the adenoviral vector application. Unlike Tenon's capsule and sclera, the subcellular localization of CDKN1B in corneal fibroblasts was almost undetectable, in both the nucleus and the cytoplasm. Thus, our results confirm the CDKN1B-specific inhibition of fibroproliferation in situ after sclerectomy. 

It is currently clear that the deficiency of CDKN1B may induce Cdk2 and Cdk4 activity when cells reenter the cell cycle. In the present study, Ad-CDKN1B–induced expression of CDKN1B is accompanied by inhibition of Cdk2 and Cdk4 activity, and leads to the cell-cycle arrest in the G₁-phase. ^40–43 This is consistent with the ability of CDKN1B to bind to and form ternary complexes with cyclin-Cdks. The interaction between CDKN1B and the cyclin-Cdk complexes inhibits the activities of G₁/S- and S-phase Cdks, including cyclin E/Cdk2, cyclin A/Cdk2, and cyclin D1/Cdk4, ^34,40 which inhibits cell-cycle arrest due to the very low or no phosphorylation of the pRb protein. ⁴⁴ The cyclin-Cdk complexes, which are active within the nucleus, sometimes can act in both the nucleus and the cytoplasm. The nucleocytoplasmic shuttling means that they directly phosphorylate in both the nucleus and the cytoplasm. ^44,45 Our observation reveals weak or moderate nuclear and cytoplasmic immunoreactivity to Cdk2 and Cdk4 induced by Ad-CDKN1B, suggesting that CDKN1B inhibits the nuclear and cytoplasmic expression of Cdk2 and Cdk4, thereby arresting DNA and centrosome duplication. 

PCNA is used to determine the positive number of fibroblasts that are actively proliferating (i.e., growth fraction and S-phase index). ⁴⁶ Our data suggest a lower score of positive cells of PCNA relative to CDKN1B throughout the experimental period. This reduction in nuclear immunoreactivity indicates the inhibition on proliferation and repair mechanics. The Cip/Kip family, including CDKN1A and CDKN1B, share a common N-terminal domain for binding to cyclin-Cdk complexes. The CDKN1A also binds to PCNA through a separate C-terminal domain affecting DNA replication and repair, thereby forming cyclin-Cdk-CDKN1A-PCNA quaternary complexes. ⁴⁰ The CDKN1B also contains unique C-terminal domains whose functions are unknown, but shares very little primary sequence similarity with CDKN1A. 

In conclusion, this study demonstrates that the sustained overexpression of CDKN1B mediated by Ad-CDKN1B in Tenon's fibroblasts in sclerectomy rabbits leads to the inhibition of fibroblast proliferation, and the downregulation of Cdk2 and Cdk4 activity after GFS. The results indicate that the CDKN1B/Cdks pathway in the model of GFS may regulate the cellular proliferation in a coordinated manner. 

Acknowledgments

The authors thank Yu-mei Tian and Jian-xin Liu (Research Center for Neuroscience, Xi'an Jiaotong University, Xi'an, China) for technical assistance. The 293 cell line was a kind gift from Zeng-hui Teng (Fourth Military Medical University, Xi'an, China). 

Footnotes

 Supported by the Key Science and Technology Program of Shanxi Province, China Grant 2009K17‐02, and Xi'an Science and Technology Program, China Grant HM1116(5).

Footnotes

 Disclosure: J.-G. Yang, None; Y. Deng, None; L.-X. Zhou, None; X.-Y. Li, None; P.-R. Sun, None; N.-X. Sun, None