ADP-ribosylation factor as a novel target for corneal neovascularization regression

Reagents and antibodies

The ARF1 inhibitor (SC-3030) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rat anti-mouse CD31 (MEC13.3) mAbs was purchased from BD PharMingen (San Diego, CA). Alexa Fluor 488 donkey anti-rat immunoglobulin G (Ig G) and the Alexa Fluor 488 annexin V/Dead Cell Apoptosis Kit (catalog no. V13241) were provided by Invitrogen Life Technologies (Carlsbad, CA). Sodium hyaluronate (HA) and Avertin (tribromoethanol) were purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). Human retinal endothelial cells (HRECs) were purchased from Y-J Biologic (Shanghai, China).

Mice

Specific pathogen-free 6- to 7-week-old male BALB/c mice weighing 20 to 25 g were obtained from Shanghai SLAC Laboratory Animal Co. Ltd. (Shanghai, China) and were kept in our animal facility under specific pathogen-free conditions. All animal experiments were conducted in accordance with the Guideline for the Care and Use of Laboratory Animals of the Chinese Medical Academy and the Soochow University Animal Care Committee, and with the Association for Research in Vision and Ophthalmology Statement for the Use of Animals in Ophthalmic and Vision Research. Animals were kept in groups of five and fed regular laboratory chow and water ad libitum. A 12 h:12 h light-dark cycle was maintained.

Alkali-induced corneal injury model

Mice were anesthetized at about 7 to 8 weeks old with an intraperitoneal injection of 1.8% (vol/vol) Avertin at a dose of 0.15 ml/10 g bodyweight. Corneal injury was induced by placing a 2 mm² filter disc saturated with 1N NaOH onto the left eye of the mouse for 40 s, as previously described [10-14]. Alkali-injured mice were divided randomly into two groups. In the neovascularization enumeration or gene detection experiments, the alkali-treated eyes received 5 μl of ARF1 inhibitor dissolved in 0.2% sodium HA at a concentration of 25 μg/ml, or 5 μl of 0.2% sodium HA as vehicle twice a day for 2 weeks from day 7 after the alkali injury. At the indicated time intervals, the mice were killed. The corneas were removed and fixed in acetone for 20 min and then used for whole mount CD31 staining. In other experiments, the corneas were removed and placed immediately in RNAlater (Qiagen, Hilden, Germany), and kept at −86 °C until total RNA extraction was performed. Each experiment was repeated at least three times.

Biomicroscopic examination

Eyes were examined with a slit lamp from Haag-Streit (BQ 900, Bern, Switzerland) and the results were photographed 3 weeks after alkali injury. Briefly, under anesthesia, photographs of the corneas were obtained using a digital camera (Nikon, Tokyo, Japan) linked to the slit lamp.

Enumeration of corneal neovascularization

Corneal whole mount staining with CD31 was performed, and blood vessels in the corneas were measured according to previous reports [15]. The mice were killed using cervical dislocation, and the corneas were rapidly removed from the alkali-treated eyes; three to four relaxing radial incisions were made in the corneas. The corneal flat mounts were rinsed in phosphate buffered saline (PBS; 8 g NaCl, 0.2 g KCl, 1.44 g Na₂HPO₄, 0.24 g KH₂PO₄, pH 7.4), fixed in acetone, rinsed in PBS, soaked in 0.3% hydrogen peroxide solution to eliminate endogenous peroxidase, blocked in 2% donkey serum albumin, stained with rat anti-mouse CD31 (1:150; BD PharMingen) at 4 °C overnight, and washed in PBS. The corneas were then incubated with Alexa Fluor 488 donkey anti-rat immunoglobulin G (1/100) for 1 h at room temperature in the dark. Digital pictures of the flat mounts were taken, and the area covered by CD31 was measured morphometrically using NIH Image software (National Institutes of Health, Bethesda, MD). The total neovascularization area was then normalized to the total corneal area, and the percentage of the cornea covered by vessels was calculated. The relative neovascular area was compared between the treated group and the control group.

Flow cytometric analysis of apoptosis

HRECs were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal calf serum (FCS) in six-well plates at 1×10⁵ cells/well for 2 to 3 days in a humidified incubator at 37 °C in 5% CO₂. After 24 h serum starvation, HRECs were cultured for 24 h in Dulbecco’s modified Eagle’s medium containing 10% FCS alone or with ARF1 inhibitor at a concentration of 14 μM, as previously reported [16]. Thereafter, cells were harvested and stained with the Dead Cell Apoptosis Kit with Annexin V Alexa Fluor 488 and propidium iodide dye; the cell apoptosis ratio was measured using a flow cytometer.

Semiquantitative reverse-transcription polymerase chain reaction

Total RNAs were extracted from the corneas with the RNeasy Mini Kit (Qiagen, Tokyo, Japan) according to the manufacturer’s instructions. The resultant RNA preparations were further treated with RNase-Free DNase (DNase) I (Life Technologies Inc., Gaithersburg, MD) to remove genomic DNA. Two micrograms of total RNA were reverse-transcribed at 42 °C for 1 h in 20 μl of reaction mixture containing mouse Moloney leukemia virus reverse transcriptase and hexanucleotide random primers (Qiagen). Serially twofold-diluted cDNA was amplified for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) to estimate the amount of transcribed cDNA. Then, equal amounts of cDNA products were amplified for the target genes using the primers under the following conditions: denaturation at 94 °C for 2 min, followed by the optimal cycles of 30 s at 94 °C, 30 s at 56–58 °C, 35 s at 72 °C, and a final 8 min extension step at 72 °C. The primers and PCR conditions used are shown in Table 1. Ten microliters of the amplified PCR products were fractionated on a 2.0% agarose gel and visualized with ethidium bromide staining. The band intensities were measured, and their ratios to GAPDH were determined with NIH Image Analysis software.

Statistical analysis

The means and standard error of the mean (SEM) were calculated for all parameters determined in the study. Values were processed for statistical analyses (Student t test) with statistical software SPSS 15.0 (SPSS Inc., Chicago, IL) p<0.05 was considered statistically significant.

Intracorneal ADP-ribosylation factor 1 mRNA expression after alkali injury

ARF1 mRNA expression in corneas after alkali injury was examined with reverse-transcription PCR (RT–PCR). ARF1 mRNA was faintly detected in the normal corneas, but was markedly increased in the injured corneas at day 7, 14, and 21 after alkali injury, and reached a peak on the 14th day (Figure 1A, B).

Effect of ARF1 inhibitor on alkali-induced corneal neovascularization

The effect of the ARF1 inhibitor on alkali-induced CNV was explored. In the alkali-induced CNV model, CNV was formed at 1 week after the alkali injury. From day 7 after alkali injury, the mice were locally administered 25 μg/ml of the ARF1 inhibitor in 0.2% HA or 0.2% HA as vehicle twice per day for 2 weeks. Microobservation concerning the CNV 3 weeks after alkali burn showed that CNV had significantly decreased in the ARF1 inhibitor–treated group compared to the control group (Figure 2A).

To further observe the effect of the ARF1 inhibitor on CNV, corneal whole mount staining by CD31—a neovascular marker—was performed. The ARF1 inhibitor–treated group showed a small number of thinner new blood vessels compared to the control group. The CNV areas in the treated group were significantly smaller than those of the control group (p<0.05), suggesting that the ARF1 inhibitor induced regression of newly formed vessels in the corneas (Figure 2B, C).

Enhanced intracorneal caspase-3 and reduced vascular endothelial growth factor messenger ribonucleic acid expression after ARF1 inhibitor intervention

Vascular endothelial cell proliferation or apoptosis is a critical step in angiogenesis. Vascular endothelial growth factor (VEGF) is a major mediator of CNV, and caspase-3 is the main factor in apoptosis. Caspase-3 and VEGF expression was detected with RT–PCR at 1 and 2 weeks after ARF1 inhibitor intervention. The results showed that the ARF1 inhibitor–treated group exhibited significantly increased intracorneal caspase-3 mRNA expression and decreased intracorneal VEGF mRNA expression compared with the control group (p<0.05), suggesting that the ARF1 inhibitor upregulated caspase-3 expression and downregulated VEGF expression in the corneas after alkali burn (Figure 3A-C).

ADP-ribosylation factor 1 inhibitor induced apoptosis in human retinal endothelial cells

To determine whether the effect of the ARF1 inhibitor on CNV is caused by endothelial cell apoptosis, ARF1 expression was detected with RT–PCR performed using the total RNA from the cultured HRECs (Figure 4A). The effect of the ARF1 inhibitor on the HRECs was further determined with flow cytometry. HRECs were exposed to Dulbecco’s modified Eagle’s medium containing 10% FCS alone in the presence or absence of 14 μM of the ARF1 inhibitor. After 24 h, the ratio of apoptosis cells (Figure 4B) in the ARF1 inhibitor–treated group (17.34±1.61%) was significantly increased compared to the control group (6.88±1.18%; p<0.05), suggesting that the ARF1 inhibitor promoted vascular endothelial cell apoptosis and then induced CNV regression.