Atorvastatin, purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany), was dissolved in DMSO (Sigma-Aldrich; Merck KGaA) at 0.05 mol/l as a stock. The perfusion pump system was purchased from Harvard Bioscience (Holliston, MA, USA). Fluorescein isothiocyanate (FITC)-phalloidin (catalogue number: 40735ES75) was purchased from Yeasen (Shanghai, China). Anti-vinculin antibody (catalogue number: BM4051) and anti-β-catenin antibody (catalogue number: BM3905) were purchased from Boster Biological Technology (Wuhan, China). High-glucose Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were obtained from Gibco (Thermo Fisher Scientific, Inc., Waltham, MA, USA). A Cell Counting Kit-8 (CCK-8) was obtained from Dojindo Molecular Technologies, Inc. (Kumomoto, Japan). Oligonucleotide primers for reverse-transcription quantitative polymerase chain reaction (RT-qPCR) were designed and synthesized by BioTNT Corp. (Shanghai, China).

A total of 90 fresh enucleated porcine eyes from 4 to 5-month-old pigs were obtained from Jiading Town Abattoir (Shanghai, China) within 4 h after death for perfusion and TM-cell isolation. Atorvastatin stock solution was diluted with Dulbecco's PBS containing 5.5 mM glucose (GPBS). The eyes were randomly divided into five treatment groups (n=6 each): Atorvastatin (17, 50, 100 and 200 µM) containing dimethyl sulfoxide (DMSO) (0.38%), plus GPBS containing the same concentration of DMSO as the control group. Animal procedures were in compliance with the Statement for the Use of Animals in Ophthalmic and Vision Research by the US Association for Research in Vision and Ophthalmology. The protocol was approved by the Institutional Animal Use and Care Committee in Fudan University.

Perfusion of enucleated porcine eyes was performed as previously reported (35). Fresh porcine eyes were cleaned off extraocular tissue and the posterior segment was submerged to the limbus in PBS at 34°C. A 21-gauge infusion needle was inserted through the peripheral cornea into the anterior chamber. This needle was carefully threaded through the pupil and the needle tip was positioned within the posterior chamber to prevent deepening of the anterior chamber that would have otherwise led to an artificial increase in outflow facility. The infusion needle was connected to polyethylene tubing, which was connected via a 3-way valve to i) a drug (or control) solution reservoir elevated relative to the eye to generate a 15-mmHg hydrostatic pressure, and ii) a syringe containing perfusion solution (drug or GPBS) on a perfusion pump, and simultaneously a pressure sensor in parallel. The IOP in the system as monitored by the pressure sensor was recorded using a computer. The perfusion rate was regulated by adjusting the perfusion pump to maintain a constant pressure of 15 mmHg. The computer also recorded the perfusion rate (F in µl/min). In addition, a second needle was inserted intracamerally into the anterior chamber and connected to a fluid collection chamber. During baseline perfusion, connections to the (drug or control solution) perfusion pump and the second (collection) needle were closed. All eyes were perfused for 30 min at a constant pressure of 15 mmHg and the initial baseline aqueous outflow facility (C₁) was recorded. The C value was obtained by dividing the perfusion flow rate by the corresponding intraocular pressure (C₁=F/IOP at baseline) as described previously (3). F and IOP were constantly measured at 10 Hz. C was calculated by averaging the values over a 10-sec window and electronically recorded every 10 sec by LabView version 7.1 (National Instruments, Austin, TX, USA). After 30 min, the anterior chamber content was replaced with 5 ml atorvastatin or control solution. Solution exchange was performed by stopping the perfusion pump and opening the connections to the drug (or control) solution reservoir and the second (collection) needle to allow the solution in the drug reservoir to flow into the eye by gravity. After exchange of 5 ml solution, connections to the reservoir containing the drug and the second (collection) needle were closed again and the perfusion pump restarted. A stable outflow facility was then obtained and recorded as C₂. Total time of the perfusion was controlled within 2 h. The difference value (C₂-C₁) was reported as ΔC. The percentage change from the baseline was recorded as (ΔC/C₁).

Primary porcine TM cells were pooled from fresh porcine eyes (n=20) via the tissue adherence method (36). TM cells were identified by immunofluorescence analysis of the presence of fibronectin, laminin and vimentin. TM cells were cultured in high-glucose DMEM supplemented with 10% FBS, 10,000 U/ml streptomycin and 10,000 U/ml penicillin. Cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ and cells of the passages 3–5 were used in the present study.

Fresh solutions of 10, 25, 50, 100 and 200 µM atorvastatin were prepared in culture medium, whereas the control group was treated with vehicle only. The final concentration of DMSO was kept below 0.5% in the culture medium.

TM cells were cultured to confluence on 2% gelatin-coated glass coverslips and treated with atorvastatin (10–200 µM) or vehicle for 24 h. Cellular morphological changes were recorded with a phase-contrast microscope (ECLIPSE Ni-U; Nikon, Tokyo, Japan). Following drug treatment for 24 h, the cells were fixed with 4% paraformaldehyde and permeabilized with 0.3% Triton X-100 in PBS, and subsequently blocked in 10% bovine serum albumin (Sigma-Aldrich; Merck KGaA) for 1 h. The cells were labelled with FITC-phalloidin (1:200 dilution) and primary antibody against vinculin or β-catenin (both 1:100 dilution) for 2 h at room temperature. After incubation with tetramethylrhodamine-labeled secondary antibody (Jackson ImmunoResearch Labs; West Grove, PA, USA; catalogue number: 111-025-003, 1:100 dilution) for 1 h at room temperature, coverslips were stained with DAPI for 1 min and observed with a fluorescence microscope (Nikon).

CCK-8 was used to evaluate the effect of atorvastatin on the viability of TM cells. At 6, 12, 24 and 48 h after treatment with atorvastatin (50–200 µM) or vehicle, 10 µl CCK-8 solution was added to 100 µl TM cells per well (~3×10³ cells) in 96-well plates, cells were maintained at 37°C in a humidified atmosphere containing 5% CO₂ for 2 h. Optical density (OD) at 450 nm was then measured using a microplate reader (BioTek, Winooski, VT, USA). The OD value was used for quantification of cell proliferation at the different dosages of atorvastatin as well as different time-points, following the manufacturer's protocol.

To determine the effect of atorvastatin on the expression of vinculin and β-catenin, total RNA from atorvastatin (50–200 µM) or vehicle-treated 0–48 h TM cells was isolated using TRIzol reagent (Invitrogen; Thermo Fisher Scientific, Inc.) and reverse-transcribed to complementary (c)DNA using a First-Strand cDNA synthesis kit (BioTNT, Shanghai, China; catalogue number: A2010B0B01) according to the manufacturer's protocol. The cDNA was then amplified by PCR using sequence-specific forward and reverse oligonucleotide primers: Vinculin, forward 5′-ACCAGGCTCCCAAGACCCAT-3′ and reverse 5′-CAGGCGAGTCAGCAGCAACA-3′; β-catenin, forward 5′-GAGGACAAGCCCCAGGATTA-3′ and reverse 5′-AGCAGTCTCATTCCAAGCCA-3′; and GAPDH, forward 5′-CGGAGTCAACGGATTTGGTCGTAT-3′ and reverse 5′-AGCCTTCTCCATGGTGGTGAAGAC-3′. A real time qPCR kit (BioTNT, Shanghai, China; catalogue number: A2010A001) was used according to the manufacturer's protocol to create the PCR reaction mixtures composed of buffers, dNTP, heat activated Taq DNA polymerase mixtures, MgCl₂ solution and premixed SYBR GREEN dyes. Amplification was performed at 95°C, 5 min; 95°C, 5 sec, 60°C, 30 sec for 40 cycles. mRNA expression was analyzed on the ABI 7500 qRT-PCR System (Applied Biosystems; Thermo Fisher Scientific, Inc.) (37).

Lysates of TM cells treated with atorvastatin (50–200 µM) or vehicle for 0–48 h were prepared using radioimmunoprecipitation assay buffer (Beyotime Institute of Biotechnology, Shanghai, China) containing phenylmethylsulfonyl fluoride, a protease inhibitor. Protein concentration was determined using the BCA assay (Yeasen, Shanghai, China; catalogue number: 20201ES76) according to the manufacturer's protocol using bovine serum albumin as a standard. Protein extracts (20 µg protein/lane) were separated by SDS-PAGE (10% acrylamide) and transferred onto a polyvinylidene fluoride membrane (EMD Millipore; Billerica, MA, USA). The membrane was then probed overnight using antibodies specifically directed against vinculin (Boster Biological Technology, Ltd., Wuhan, China; BM4051, 1:100 dilution), β-catenin (Boster Biological Technology, Ltd., BM3905, 1:100 dilution) and GAPDH (Cell Signaling Technology, Inc., Danvers, MA, USA; catalogue number: 2118; 1:1,000 dilution) at 4°C, followed by incubation with horseradish peroxidase-conjugated goat anti-rabbit secondary antibody (Solarbio, Beijing, China; catalogue number: SE134; 1:1,000 dilution) at room temperature for 1 h. Specific bands were visualized by Odyssey infrared imaging system (LI-COR, Inc., Lincoln, NE, USA). The signals were analyzed using ImageJ software (version 1.48v; National Institutes of Health, Bethesda, MD, USA). The band densities of each sample were normalized to the respective GAPDH band. All results were repeated three times.

To evaluate whether the atorvastatin-induced effect is reversible, the medium of TM cells treated with atorvastatin (100 µM) for 48 h was replaced with culture medium after washing with PBS. The reversibility of cellular morphological changes was observed using a phase-contrast microscope and images were captured. At 24 h after drug washout, cells were fixed and stained for F-actin, vinculin and β-catenin using the same procedures described in Atorvastatin treatment and cytoskeletal staining section.

Data were analyzed using STATA version 12.0 software (StataCorp LLC, College Station, TX, USA). Values are expressed as the mean ± standard error. One-way analysis of variance followed by Bonferroni's multiple comparisons test was used for statistical comparisons of multiple groups. P<0.05 was considered to indicate a statistically significant difference.

As expected, there was no significant difference baseline outflow facility (C₁) values among all treatment groups (Fig. 1A). There was no significant difference in outflow facility between the control and 17 µM atorvastatin-treated group. Concentrations of atorvastatin of ≥50 µM significantly increased the outflow facility. C₂ value in the 50, 100 and 200 µM groups was significantly increased compared with C₁ value in each group (6.96-, 9.59- and 6.99-fold increase, respectively; Fig. 1A). There was no significant difference among the three higher-concentration groups (Fig. 1B).

Treatment of confluent TM cells with atorvastatin (10–200 µM) for 24 h led to certain morphological changes. Compared with the control group, no obvious morphological changes were detected in TM cells treated with 10 µM atorvastatin. However, cells treated with ³25 µM atorvastatin exhibited progressive cell rounding and separation among cells, while the cells did not detach from the culture dish (Fig. 2). Similar results were observed in three independent experiments.

To evaluate the potential effect of atorvastatin on cell viability, porcine TM cells were treated with 50–200 µM atorvastatin and subjected to a CCK-8 assay at 6, 12, 24 and 48 h. Compared with that in the mock-treated group, no significant change in cell viability was detected after treatment with atorvastatin (50–100 µM) for 48 h. However, the cell viability indicated a downtrend in the group treated with 200 µM atorvastatin after 24 h; although this was not significantly different. (Fig. 3). Therefore, these findings suggest that high concentrations of atorvastatin may inhibit TM cell viability. And experimental results may be statistically significant after extending the intervention time.

From Fig. 4, it may be observed that the distribution of F-actin (green) in the mock-TM cells was relatively uniform, while vinculin (red) and β-catenin (red) were mainly distributed around the nuclei. A dose-dependent, marked reduction of F-actin and changes in the distribution of vinculin and β-catenin were observed in TM cells following 24 h of treatment with atorvastatin (10–200 µM). An association between changes in the actin cytoskeletal organization/distribution of focal adhesions and morphological changes in TM cells was apparent (Fig. 4).

To confirm the results obtained by immunohistochemistry, RT-qPCR and western blot analysis were performed. Compared with that in control cells, the expression of vinculin and β-catenin mRNA was significantly decreased in TM cells treated with 100 µM atorvastatin for durations of ≥12 h (Fig. 5A and B). Similar results were obtained by western blot analysis, which demonstrated that the expression of vinculin and β-catenin protein was decreased by atorvastatin (Fig. 5C and D).

Of note, the atorvastatin-induced morphological changes, modification of F-actin organization and focal adhesions were reverted to those prior to treatment within 24 h of replacing the culture supernatant with atorvastatin-free medium (Fig. 6).