The effect of phosphorylated Akt inhibition on posterior capsule opacification in an ex vivo canine model

Samples

Normal eyes were obtained by enucleation from dogs in good general health that were humanely euthanized at a local animal shelter for population control purposes. The eyes were examined with a focal light source and deemed free of corneal disease, cataract, or other anterior segment disease. All dogs used in this study were estimated to be between 1 and 8 years of age, based on dentition and thickness of the anterior lens capsule. Globes were collected within one hour of death and placed in dilute betadine solution, then rinsed and immersed in 1× phosphate buffered saline solution (PBS, pH 7.2) until dissection. Excess conjunctiva and Tenon’s capsule were excised from the perilimbal region, and a stab incision was made into the anterior chamber 1 mm posterior to the limbus. Anterior lens capsules with adherent LEC were excised from each lens for protein extraction and immediately frozen and stored at −70 °C until extraction. Whole lenses and anterior lens capsules with adherent LEC were also fixed in 10% neutral-buffered formalin for immunohistochemical staining.

Cataractous lens capsules were obtained from clinically normal dogs undergoing elective phacoemulsification cataract surgery at The Ohio State University College of Veterinary Medicine. These capsules consisted of mature and hypermature cataracts that were considered either diabetic or non-diabetic in their etiology. These canine cataracts almost universally have LEC that have undergone EMT and posterior migration onto the posterior capsule [32]. Samples chosen for this study had obvious subcapsular plaques or diffuse LEC proliferation onto the posterior lens, evident at the time of surgery. Patients with only nuclear cataract were not included in this study. Cataractous samples were obtained by continuous curvilinear capsulorhexis and either immediately placed in 10% neutral-buffered formalin or snap frozen and stored at −70 °C until protein extraction.

All fixed samples were paraffin embedded, sections, stained with hematoxyline and eosin (H&E), and then examined by light microscopy. For immunohistochemical staining, samples were sectioned onto charged slides (ProbeOn Plus; Fisher Scientific, Pittsburg, PA).

Primary cultures

Anterior lens capsules with adherent LEC were obtained via dissection from normal canine eyes, as described above. Following dissection capsules were incubated in trypsin (0.25% trypsin and 1× EDTA; Gibco, Carlsbad, CA) for 5 min at 37 °C. After incubation, the solution and lens capsule were centrifuged for 2 min at 0.3× g. Fluid was decanted and supplemented DMEM (containing 10% fetal bovine serum and 1% antibiotic/antimycotic [Gibco]) was then added. The solution, including the lens capsule, was transferred to a laminin-coated culture flask (Beckton-Dickinson, Franklin Lakes, NJ) an incubated in a humidified incubator at 37 °C and 5% CO₂. LEC were grown until 90% confluence before re-plating.

Irradiation and treatment with AR-12

Cultures of primary LEC were washed and covered with PBS, then exposed to UV irradiation. The UV source was from Philips TL20 W/12 RS UV-B lamps (American Ultraviolet Company, Murray Hill, NJ). These lights emit wavelengths of light between 280 and 400 nm, with a peak at 313 nm. The lamps emit approximately 60% UV-B and 40% UV-A [34]. Radiation was filtered through Kodacel (Eastman Kodak, Rochester, NY), to remove wavelengths below 280 nm [35]. Based on preliminary and previously published data, the cells were irradiated with either 0 or 600 J/m², which can induce expression of both pAkt and telomerase [23,27]. Following exposure, PBS was removed and serum-free DMEM containing AR-12 was placed on the cells. The doses of AR-12 used were 0 (vehicle only), 2.5, 5.0, and 10.0 µM (n=6). Cells were collected 8 or 24 h after irradiation for immunoblot and telomerase activity analysis.

Ex vivo capsules

An extracapsular cataract extraction with lens capsule dissection was performed as described previously [36-38]. Residual cortical material was removed gently by manual irrigation/aspiration with a coaxial cannula, but no effort was made to polish the anterior or posterior lens capsule. The lens capsule was excised from the zonular and vitreal attachments and pinned to a polymethylmethacrylate (PMMA) Petri dish also as previously described [38]. All capsules were cultured in a total of 5.0 ml of DMEM containing 1% antibiotic and antimycotic (Gibco).

Treatment groups

Each group contained 8 lens capsules and 5 groups were treated with 0.0, 2.5, 5.0, 7.5, or 10.0 μM AR-12, which was synthesized in-house as described previously [26]. The lens capsules were incubated at 37 °C in 5% CO₂, and the culture medium replaced every 3 days for 14 days. Morphologic observations were made with phase-contrast microscopy. Two additional groups of lens capsules were harvested (n=6 per group). One group was exposed to DMEM supplemented with 10.0 μM AR-12 for the first week of incubation, followed by a week of DMEM alone. The second group was exposed to DMEM alone for the first week of incubation, followed by DMEM supplemented with 10.0 μM AR-12 for the second week of incubation. After 2 weeks, all lens capsules were fixed in 10% neutral-buffered formalin, embedded in paraffin, and sectioned.

Telomeric repeat amplification protocol (TRAP)

The TRAP assay, modified by the Intergen Company (Norcross, GA), is an extension of the original method [18]. In the first step, telomerase, if present in the protein lysate, adds several telomeric repeats onto the 3′ end of a biotinylated telomerase substrate oligonucleotide (b-TS). In the second step, the extended products are amplified by PCR using Taq polymerase, the b-TS and RP (reverse) primers, and a deoxynucleotide mix containing dCTP labeled with dinitrophenol, generating a ladder of products with six base increments, starting at 50 nucleotides (Chemicon, Temecula, CA). Briefly, for our studies, a master mix was prepared with 5× TRAP Reaction mix, Taq polymerase (Panvera Takara, Carlsbad, CA), and RNase-free water. One µg of protein was added to the Master mix for a final volume of 50 µl; all set-up steps were performed on ice. Primer extension was then performed at 30 °C for 30 min, followed by 33 cycles of amplification as follows: denaturation for 30 s at 94 °C and annealing for 30 s at 53 °C. Samples were held at 4 °C until detection using hybridization and ELISA (absorbance measured at 450 nm with reference wavelength of 690 nm) following manufacturer’s protocol, and electrophoresis on a 10% nondenaturing polyacrylamide gel that was subsequently stained with SYBR gold (1:10,000; Molecular Probes, Carlsbad, CA). By comparing the PAGE gel method of evaluating TRAP results with the ELISA methods, we have set 0.100 units as the lowest amount of detectable telomerase activity.

Histological and immunohistological evaluation

Routine H&E staining was performed on ex vivo capsules to evaluate cell morphology and cell counts. All LEC adhered to the capsule were manually counted in 10 H&E sections per capsule. The quantitative analysis was performed on sections taken as close as possible to the capsule midline. Cell counts were performed by two independent observers.

Immunohistochemical staining was performed as previously described [38] using antibodies against TERT (1:350; Calbiochem, San Diego CA), pAkt (1:350; Santa Cruz, Santa Cruz, CA), cleaved caspase-3 (1:20; Oncogene, San Diego CA), ILK (1:200; Stressgen, San Diego CA), α-SMA (1:100; Spring Bioscience, Freemont CA), and PCNA (1:200; DAKO Corp., Carpinteria CA). Secondary antibodies included, as appropriate, anti-goat (Vector Laboratories, Burlingame CA), anti-rabbit (Biogenex Super Sensitive Link; Biogenex, San Ramon CA), and anti-mouse (Vector Laboratories) antibodies. DAKO Antibody Diluent with Background Reducing Components (DAKO) was used to dilute antibodies. Immunostaining was visualized with either diaminobenzidine tetrahydrochloride (DAB, DAKO) applied for 3 min at room temperature in the dark or with Vector NovaRED (Vector Laboratories) applied for 5 min at room temperature.

Appropriate positive and negative controls were performed with each experiment. Canine squamous cell carcinomas were used as a positive control for TERT, pAkt, α-SMA, ILK, and caspase-3. A normal canine lymph node was used as a positive control for PCNA. Negative controls omitted the primary antibody.

Immunobloting

Whole cell protein was extracted from frozen positive controls, normal canine anterior lens capsules, and anterior capsulotomy samples from clinical canine cataracts using 1× CHAPs lysis buffer (Chemicon, Temecula, CA) in accordance with the manufacturer’s instructions with minor adaptations for our tissue as previously described [19]. Protein concentration was quantified using the Bradford protein assay (Bio-Rad, Hercules, CA).

Samples were denatured (95 °C, 3 min) in modified sodium dodecyl sulfate (SDS) sample loading buffer, and protein (15 µg) was separated by SDS/PAGE (10% acrylamide, v/v) at 150 V for approximately 1.5 h. After electrophoresis, proteins were transferred to a nitrocellulose membrane at 300 mA for 1.5 h. Nonspecific binding was blocked by incubating the membrane for 5 h at room temperature in 5% nonfat dry milk diluted in 1× Tris-buffered saline containing 0.1% Tween-20 (TBST), After blocking, membranes were incubated overnight with pAkt (Cell Signaling, Danvers, MA), diluted 1:500 in blocking buffer. The membrane was then washed extensively with 1× TBST, and secondary antibody (donkey anti-rabbit, Santa Cruz) was added in blocking buffer (1:8,000) for 1 h at room temperature. Protein was detected using the Pierce (Rockford, IL) Femto Immunoblotting system. Membranes were stripped (Pierce) and the technique was repeated using anti-β-actin antibody (1:5,000; Sigma-Aldrich, St. Louis, MO). Kodak 1D Image Analysis Software (Kodak Molecular Imaging, New Haven, CT) was used to obtain densitometry readings for all western blots.

Statistical analysis

All statistical analysis was performed with Prism version 4.0 software (GraphPad Software Inc. San Diego, CA). All data was analyzed using one-way ANOVA (ANOVA) with Tukey’s post-test. The capsule cell counts were evaluated with a non-paired Students’ t-test. The level for statistical significance was set at p<0.05 for all comparisons. All graphs were generated in Prism version 4 and display the mean and standard error of the mean (SEM), represented by error bars.

pAkt expression in normal and cataractous lens epithelial cells

As evaluated by immunohistochemisty, normal canine LEC had minimal to no detectable pAkt protein present (Figure 1A), while immunostaining for pAkt was strong predominantly in the cytoplasm of naturally occurring cataractous canine LEC (Figure 1B) and clinical samples of canine PCO (Figure 1C). Western immunoblotting confirmed that LEC from clinical cataract samples had increased expression of pAkt protein compared to normal LEC (Figure 1D). Samples from our ex vivo model of PCO formation demonstrated strong expression of pAkt, as observed by western blot (Figure 1D).

Expression of pAkt and telomerase activity follow irradiation and AR-12 treatment

Irradiation of LEC significantly induced expression of pAkt at both 8 (p<0.05; Figure 2A) and 24 (p<0.01; Figure 2B) h compared to controls, as observed by western blot analysis. In the presence of AR-12, all doses tested were capable of significantly (p<0.01) decreasing pAkt expression in irradiated LEC compared to LEC that received UV irradiation in the absence of AR-12 (Figure 2A,B). This was observed at both 8 (Figure 2A) and 24 (Figure 2B) h. Cells treated with AR-12 alone did not significantly alter pAkt expression (Figure 2A,). As previously demonstrated [27], when assessed by the TRAP assay, telomerase activity was significantly (p<0.05) increased in LEC that were exposed to irradiation and allowed to recover for 24 h (Figure 2C). When UV-exposed LEC were allowed to recover in the presence of either 5.0 or 10.0 µM AR-12, there was a significant (p<0.05) decrease in telomerase activity compared to the cells that were irradiated and received 0.0 µM AR-12 (Figure 2C).

Control ex vivo lens capsules

Immediately post-dissection, lens capsules had visible LEC present on the anterior capsule only. By 3 days post-dissection, untreated LEC had migrated across the anterior and posterior lens capsules. By 7 days post-dissection, LEC were present on the posterior capsule in the central visual axis in most lens capsules, and all had reached confluence by 14 days post-extraction (Figure 3A). The LEC morphology was cuboidal to hexagonal and there was little anisocytosis. Hematoxylin and eosin staining confirmed the phase contrast findings (Figure 3B). At 2 weeks post-dissection, the control lens capsules contained confluent cells on both the anterior and posterior capsules with focal areas of LEC hyperplasia in the equatorial region.

AR-12 treated ex vivo lens capsules

All LECs in the lens capsules treated with 2.5 μM AR-12 in DMEM displayed mild intracellular vacuolization and anisocytosis. Some areas of lens capsule contained sparse LEC, but confluence was visible by 7 days after sham cataract surgery. Histopathologically, LEC were flattened and visibly fewer in numbers compared to control lens capsules (Figure 3C,D).

All lens capsules treated with 5 μM of AR-12 in DMEM had areas devoid of LECs and confluence was not achieved by 14 days after sham cataract surgery. LEC were severely vacuolated and varied in size and shape. Histologically, most areas of the anterior and posterior lens capsules were devoid of LEC; cells, as observed with phase contrast, indicated abnormal morphology and vacuolization (Figure 3E,F).

Neither lens capsule groups treated with 7.5 nor 10 μM AR-12 in DMEM supported LEC migration and proliferation. The lens capsules contained minimal numbers of LEC on phase contrast evaluation of 7.5 μM-treated capsules, and capsules were devoid of all LEC on phase contrast evaluation of 10 μM AR-12-treated. These findings were confirmed with histopathology (Figure 3G-J).

Effects of late Akt inhibition on LEC

Lens capsules treated with DMEM only for 7 days, followed by 7 days of treatment with 10 μM AR-12 in DMEM were grossly and histologically similar to the control lens capsules. By 7 days post dissection, confluence was attained across the entire posterior capsule in most lens capsules (data not shown).

Effects of early Akt inhibition of LEC

Lens capsules treated with 10 μM AR-12 in DMEM for 7 days, then treated with compound-free media for 7 days were grossly and histologically similar to those lens capsules treated with 10 μM AR-12 continuously for 14 days. The lens capsules were devoid of all LEC on phase contrast evaluation (data not shown).

Telomerase activity

All capsules with LEC present underwent TRAP evaluation for the presence or absence of telomerase activity. There was a significant decrease in telomerase activity in the AR-12 treated groups compared to the untreated controls (Figure 4). Capsules in the 10 µM treatment group did not undergo TRAP evaluation because there were no LEC were present following 14 days of culture.

Lens epithelial cell counts

The posterior capsule LEC counts of the 5.0, 7.5, and 10.0 uM -treated capsules were significantly lower than those of the control capsules (p<0.05 and p<0.01; Figure 5A). Similar results were obtained by two independent observers. The complete capsule LEC count of the 5.0 uM AR-12 was lower than those of the control capsules, however, this was not a significant difference (Figure 5B). The complete capsule LEC count of the 7.5 and 10.0 uM treated capsules were significantly lower than the complete control capsule LEC counts (p<0.01 and p<0.001; Figure 5B).

Immunohistochemical staining following pAkt inhibition

Immunohistochemical staining of LEC in all lens capsules, in all treatment groups, excluding 10 µM due to a lack of cells, showed equivalent moderate immunoreactivity for ILK, α-SMA, and PCNA (data not shown). There was positive immunoreactivity for TERT and pAkt in all treatment groups, but a subjective decrease in staining was observed in the 7.5 μM-treated groups, in comparison to untreated control, 2.5 and 5 μM-treated groups (Figure 6 and Figure 7). Caspase-3 immunoreactivity was variable, but increased numbers of positive cells were seen in lens capsules treated with AR-12 concentrations at 5 μM and above (Figure 8).