Role of bone morphogenetic proteins in form-deprivation myopia sclera

Animals

All experiments undertaken conformed to the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and the United States NIH document “Guide for the Care and Use of Laboratory Animals 1996” were followed. Twenty pigmented guinea pigs (approximately 10~15 days old) were reared as previously reported [18]. Animals were housed in groups in a temperature controlled room (25 °C) in open topped cages, on a 12 h light (provided by ceiling fluorescent tubes 36W) and 12 h dark cycle, with free access to food (guinea pig pellets and fresh vegetables) and water.

Experimental design and myopia induction

The animals were randomly assigned to 2 groups: diffusers (n=10) and normal controls (n=10). Diffuser-wear techniques employed in our experiments are similar to McFadden’s techniques [18]. The diffusers (white translucent hemispheric occluder with a diameter of 12 mm) were mounted on a matching plastic ring and glued to the periorbital fur of the eye for the whole two-week period of this experiment. The deprived eye in diffusers was randomly selected among animals and the fellow eye was untreated as a contralateral control.

Biometric measurements

Biometric measurements include streak retinoscopy and ultrasonography. Retinoscopy was performed in a dark room by using a streak retinoscope and trial lenses when the eyes had been fully cyclopleged with 2 drops of tropicamide for approximately 15 min. The refraction was recorded as the mean value of the horizontal and vertical meridians. Axial length of the eye was measured under topical anesthesia by an A-scan ultrasonagraph with 11 MHz which had been used by Lu et al. [19]. Ocular refraction and axial length were collected at the beginning and end of the experiment.

Tissue preparation

Human scleral tissues were excised from healthy adult human donor eyes (n=3; ages of 18, 19, and 25 years) obtained from the Eye Bank of the Affiliated Hospital of Medical College, Qingdao University, with the approval of Ethics Committee of Qingdao University (Qingdao, China) and complied with the tenets of the Declaration of Helsinki for biomedical research involving human subjects. Then each sclera was cut into equal halves along the optic disc and macula. The head of the optic nerve was discarded. One half was embedded with Optimum Cutting Temperature Compound (OTC; Sigma, St. Louis, MO), and cut into 8 μm sections at −20 °C. The other half was stored at −80 °C and used for RT–PCR. Sections were subsequently tiled onto slides (Corning Ltd, Tokyo, Japan), fixed with cool acetone for 15 min, air-dried, and kept frozen at −20 °C until use. Normal (n=10) and form deprived (n=10) guinea pigs were killed by overdoses of Chloral Hydrate. The eyeballs were removed and cut into halves about 1 mm posterior to the ora serrata on the ice plate. The anterior segment of the eye was discarded. The posterior sclera was excised by using a 6 mm-diameter trephine around the head of the optic nerve. The head of the optic nerve was discarded. The left sclera was flapped and stored at −80 °C.

Human scleral cell isolation and culturing

Human scleral tissues were excised from surgical specimens collected during treatment for retinoblastoma (n=2, ages of 1.5 and 2 years) under signed informed consent obtained from donors and was also approved by the Ethics Committee of Qingdao University (Qingdao, China) and complied with the Tenets of the Declaration of Helsinki for biomedical research involving human subjects. The scleral tissue was cut into small pieces (about 1 mm³) and cultured in 25 mm² flasks in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA)/Nutrient mixture F12 (1:1) with high glucose supplemented, 10% fetal bovine serum (FBS; Gibco) and incubated at 37 °C in a humidified incubator containing 5% CO₂. When achieve a heavy primary monolayer, the cells were trypsinized for 3 min at room temperature in 0.25% trypsin/EDTA solution in phosphate buffered saline (PBS, Gibco) and subcultured at a split ratio of 1:3 in a 25 mm² flask (Corning Ltd., Lowell, MA). The third passage of fibroblasts was used for this experiment.

Cell proliferation assays

Cell proliferation was measured by 3-(4, 5-dimethylthiazol-2-yl) −2,5- diphenyltetrazolium bromide (MTT, Gibco) assay. Cells were plated in 96 well plates (Corning Ltd) with DMEM in 10% FBS at a 4×10³/cm² density. Before treated with BMP-2, cells were synchronized by replacing the medium with serum-free medium for 24 h. Then cells were incubated with different concentrations (1 to 100 ng/ml) of BMP-2 at 37 °C for 1 to 6 successive days. This dose range of BMP-2 was commonly used in culture for in vitro studies although the normal physiologic levels are lower in bone at 1 to 2 ng/g. The medium was semi-replaced daily. On the seventh day, cells were washed twice with 10 mmol/l PBS (pH 7.2) and incubated with 0.5 mg/ml MTT at 10 μl/well for the last 4 h before the reaction was terminated with the addition of 150 μl dimethyl sulfoxide (DMSO, Sigma). The absorbance was determined at 490 nm using an enzyme linked immunosorbent assay (ELISA) reader (BIO-TEK Instruments, Winooski, VT).

Total RNA isolation, reverse transcription and polymerase chain reaction (RT–PCR)

Human sclera cells were seeded in 6-well culture plates at 4×10⁵ cells/well and cultured for 24 h. After synchronized with serum-free DMEM for another 24 h, cells were incubated in DMEM containing BMP-2 (100 ng/ml). The culture without BMP-2 acted as controls. On day 2, the culture media was changed with the same concentration of BMP-2 in each well. After 72 h incubation, cells were harvested for RNA extraction. Sclera was grinded in liquid nitrogen. Total RNA were extracted from sclera (normal human sclera n=3;normal guinea pigs sclera n=3; form deprived guinea pig sclera n=5; contralateral control sclera n=5) and cells with TRIzol reagent (TaKaRa, Dalian, China) and contaminating DNA was digested using DNase I (Promega, Madison, WI) according to the manufacturer's protocol, and quantified using ultraviolet spectrophotometry by measuring OD260 and OD280 (optical density 260/280 higher than 1.9). cDNA was synthesized with 5 µg total RNA, 2 µl random 6 mers, 0.5 µl Oligo dT Primer, 2 µl PrimeScriptTM Buffer, and 0.5µl PrimeScript^TM RT Enzyme Mix I (TaKaRa) at 37 °C for 15 min. The reaction was suspended at 85 °C for 5 s. Conditions for PCR were 95 °C for 30 s, 35 cycles of 95 °C for 5 s, 55 °C (56 °C for BMP-3, BMPR-lB, and Smad1, respectively) for 30 s, and 72 °C for 30 s. The final extension step was at 72 °C for 7 min. The PCR products were then electrophoresed on a 2% agarose gel containing 10× Gelred reagents in parallel with 50 bp DNA markers. The nucleotide sequences of the primers used in the experiments are denoted in Table 1. β-actin (Actb) was used as an internal control. All band intensities were evaluated by densitometry (Gel-Pro Analyzer 4.5; Gel-Pro, Bethesda, MD). The ratio of a target gene versus Actb was computed as a series of numbers where there might be the smallest number. To make the expression trend clear, the value in the figures was the ratio of the series of numbers versus the smallest one. Because guinea pigs nucleotide sequences haven’t been reported, we designed probes to cover regions where the human and rat BMP sequences show high homology. It is therefore reasonable to expect that our primers specifically recognize the examined BMPs. To prove the identity, all the experiments were performed at least 3 times and the products were sequenced by TaKaRa.

Western blot analysis

Human sclera cells were treated as described in RNA extraction. After incubated with 100 ng/ml BMP-2 for 72 h, cells were harvested for protein extraction. Form deprived guinea pig sclera (n=5) and contralateral control sclera (n=5) were grinded in liquid nitrogen. Cells and sclera tissues were homogenized separately in ice-cold extraction buffer (0.01 M Tris-HCl at pH 7.4, 0.15 M NaCl, 1% w/v Triton X-100, 0.1% SDS, 1% deoxycholic acid, 1mM EDTA) as well as protease inhibitors (1μM pepstatin, 1μg/ml leupeptin, and 0.2mM PMSF). After homogenization, samples were placed on ice for 30 min and centrifuged at 4 °C 12,000× g for 15 min. The supernatant was decanted and the precipitate was discarded. Protein concentrations were determined according to the BCA method using an Enhanced BCA Protein Assay Kit (Boster, Wuhan, China). Each protein sample (30 μg) was mixed with 5× sample buffer for SDS PAGE. The mixture was boiled for three min, electrophoresed on a 10% SDS polyacrylamide gel, and transferred to nitrocellulose membranes (Pall Corporation, East Hills, NY). Protein loading and transfer efficiency were monitored by staining the membranes with 1% Ponceau S. The membranes were washed three times with TBST (pH 7.6) and soaked in a blocking solution (5% w/v skim milk powder in 2.5 mM Tris-HCl and 14 mM NaCl plus 0.05% Tween-20) for 1 h at room temperature. The membranes were incubated overnight with primary antibodies at a 1:100 dilution (rabbit anti BMP-2, −4, and −5; 0.2ml; Boster, Wuhan, China) and 1:500 dilution (rabbit anti phospho-Smad1/5/8; 0.1 ml; , Millipore, Billerica, MA) at 4 °C in blocking solution. The membranes were then washed three times with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody (goat anti-rabbit) at a 1:1,000 dilution (0.4 μg/ml; Boster) for another 1 h at room temperature. The membranes were again washed three times with TBST. The reaction products were visualized with BeyoECL Plus western blotting detection reagents (Beyotime Institute of Biotechnology, Haimen, China). Images were captured with a Fuji Film LAS3000 imaging system and analyzed with MultiGauge software (Fuji Film, Tokyo, Japan). β-actin (Kang Chen, China) was used as a housekeeping protein to normalize the protein load.

Immunofluorescent staining

The slides were washed three times with PBS, nonspecific bindings were blocked by incubation in 10% (v/v) goat serum in PBS for 30 min at room temperature. Then the slides were incubated at 4 °C overnight with primary antibodies against BMP-2, −4, and-5 (BA0585; BA0662; BA0663; 1:200 dilution; Boster) diluted in PBS. The antibody-treated and negative control slides were washed with PBS three times at room temperature, then incubated with FITC labeled goat anti-rabbit IgG antibodies (BA1105; Boster) diluted in PBS at room temperature for 1 h. Fluorescent signals were detected using a fluorescent microscope (TS-100; Nikon, Tokyo, Japan).

Cells grown on 96-well plates were fixed with 4% (v/v) paraformaldehyde for 30 min and then made permeable with 0.1% Triton X-100 in PBS for 10 min. Nonspecific binding was blocked by incubation in 10% (v/v) goat serum in PBS for 30 min at room temperature. Following incubation of the cells with primary antibody against phospho-Smad1/5/8 (AB3848; Millipore), α-SMA (BM0002; Boster) diluted in PBS at 4 °C overnight. Cells were then probed with FITC-labeled (BA1105; Boster) or Cy3-labeled goat anti-rabbit (BA1032; Boster) secondary antibodies diluted in PBS and incubated at the room temperature for another 1 h. Nuclear staining with DAPI was applied. Fluorescent signals were detected using a fluorescent microscope (TS-100; Nikon).

Statistical analysis

Data were expressed as mean±standard deviation (SD). Differences between the groups were compared by using a one-way ANOVA (ANOVA) with a Tukey post hoc test or Bonferroni Correction. The Student’s t-test was performed for statistical analysis of cytometry between the experiment and control groups. Statistical analysis was performed using the SPSS 15.0 statistical software (IBM, Chicago, IL). The results were considered to be statistically significant at p<0.05.

Expression of BMPs and BMP receptors in human and guinea pig sclera

In normal human and guinea pig sclera, BMP-2, −4, −5, and all the BMPRs could be detected after RT–PCR (Figure 1A). The relative expression level of BMP-5 was strongest, followed by BMP-2 and BMP-4. The expression of BMPR-IA and BMPR-IB were higher than that of BMPR-II. The expression of BMPs in human sclera were further confirmed by immunofluorescence (Figure 1B).

The expression of BMPs in guinea pig sclera of form-deprivation myopia model

The differences between the two eyes of each animals in axial length were not significant for the two groups at the beginning (p=0.794, one-way ANOVA with Bonferroni correction, Figure 2). At the 14th day, monocularly deprived eyes had myopia of −0.48±0.51 D and an axial length of 8.29±0.05 mm which was significantly more myopic and longer than either the contralateral control eyes (+3.22±0.34 D; t=-12.814,df=18, p<0.001; 8.05±0.06 mm, t=7.23,df=18 p<0.001) or age-matched normal eyes (+3.07±0.54 D, t=-11.878, df=18, p<0.001; 8.06±0.06 mm, t=9.084, df=18, p<0.001, independence samples test, Figure 2).

The relative expression levers of FDM eyes were significantly lower than contralateral control eyes for BMP-2 (p=0.017) and BMP-5 (p=0.028). However, the expression of BMP-4 was not significantly altered in FDM eyes (p=0.162; Figure 3A). The identity of PCR products was confirmed by sequence analysis of vector-cloned cDNA fragments. Negative control experiments without template did not yield any products. The protein expressions of BMP-2, −4 and −5 could be detected by western blot (bands at about 13 kDa and 15 kDa, Figure 3B). After 14 days of visual deprivation, significant decrease was shown for BMP-2 (−32.65%, p=0.026) and BMP-5 (−25.11%, p=0.034) compared with the contralateral control eyes of the same animals, while no significant changes shown for BMP-4 (p=0.158; Figure 3B).

The effect of BMP-2 on HSF in vitro

The scleral cells began growing out from pieces of sclera tissue after almost 10 days in culture. They exhibited a fibroblast-like spindle shape or polygonal shape in morphology for scleral cells, which grew in a vortex pattern cultured in monolayer. After exposed with various concentrations of BMP-2 (0, 1, 10, and 100 ng/ml) for one to seven days, cells proliferation was significantly higher on day 6 with 100 ng/ml BMP-2 (Figure 4A) than other concentrations, and the shape of cells was polygonal (Figure 4B,C). And there was a rapid differentiation of fibroblasts into α-SMA-expressing myofibroblasts with the addition of 100 ng/ml BMP-2 in cell cultures (Figure 4D,E).

As shown in Figure 5A,B, the expressions of aggrecan and collagen l as well as their protein expression levels were significantly increased when being cultured with 100 ng/ml BMP-2 for 72 h. The time point was selected according to Hu et al. [20] and Yoon et al. [21].

Potential signal pathway of BMP effects in vitro

The expression of BMPR-IA, -IB, -II, Samd1, and BMP-2 significantly increased in vitro (Figure 6A) after cells were exposed with100 ng/ml BMP-2 for 24 h. The expression level of BMPR-IA and BMPR-IB were higher than BMPR-II.

To confirm the effect of BMP-2 on signal pathway in vitro, cells were stained with immunofluorescence labeled Smad1/5/8 and detected by western blotting also. After incubated with100 ng/ml BMP-2 for 1 h, it showed more marked fluorescent signals localized in the nucleus of the treated cells than untreated cells (Figure 6B). Western blots also showed a higher phospho-Smad1/5/8 protein level in the treated cells (Figure 6C).