To demonstrate the effect of ZX-5 on the PI3K/Akt pathway in HRECs, the expression of Akt, Phospho-Akt (Thr473), eNOS and Phospho-eNOS (Ser1177) were tested. Compared with DMSO-treated control cells, the protein expression level of Akt, Phospho-Akt (Thr473), eNOS and Phospho-eNOS (Ser1177) were up-regulated in a concentration dependent manner in (R,R)ZX-5-treated cells, whereas (S,S)ZX-5 down-regulated the expression of Akt and had no influence on the expression of phospho-Akt (Thr473), eNOS and p-eNOS (Ser1177) (Figures 1 and 2).

PI3K/Akt signaling pathway is one of the important intracellular signal transduction pathways in response to the extracellular signals. The key effector molecule Akt in this signaling pathway exerts its effects in the cell by phosphorylating a variety of downstream substrates. The downstream targets of Akt include BAD, Caspase 9, FKHR, GLUTs, Ca²⁺ and eNOS [11]. eNOS phosphorylation at Ser1177 and Ser (Thr) 613 can enhance its activity which is affected by the PI3K/Akt.

We think that (R,R)ZX-5 up-regulates eNOS expression and increases NO release through the activation of PI3K/Akt-eNOS pathway [12]. The signal pathway transduction are as follows [13]: First, PI3K is activated, which generates phosphatidylinositol-3,4,5-triphosphate (PI3P) and phosphatidylinositol-3,4-diphosphate (PI2P). PI3P recruits PDK1 and Akt serine/threonine kinase at the plasma membrane, which results in the phosphorylation and activation of Akt. Then p-Akt is released from cellular membranes and makes eNOS phosphorylated at Ser1177 and Ser (Thr) 613. Further, p-eNOS catalytes l-arginine to generate NO (Figure 3).

The intracellular free Ca²⁺ concentration in the ZX-5 treated HRECs was detected. As (R,R)ZX-5 up-regulated the protein expression level of Akt, Phospho-Akt (Thr473), eNOS and Phospho-eNOS (Ser1177) in a concentration range of 7.5 to 30 μg/mL, a concentration of 30 μg/mL was used in this experiment. The intracellular free Ca²⁺ concentration was escalated significantly by (R,R)ZX-5 treatment, but (S,S)ZX-5 did not change the intracellular free Ca²⁺ concentration (Figure 4).

Calcium is an important and ubiquitous intracellular messenger, controlling a diverse range of cellular processes, such as gene transcription, muscle contraction and cell proliferation [14]. In response to adequate stimuli, [Ca²⁺]i (Intracellular Ca²⁺ concentration) increases, oscillates and decreases, leading to the activation, modulation and termination of cell function. Numerous channels and pumps allow this particular cation to enter and exit cells and move between the cytosol and intracellular stores. Activation of NOS requires [Ca²⁺], calmodulin, calcium-activated phosphatase, NADPH, and glutamate. eNOS (endothelial NOS) is constitutively expressed as latent enzymes and require a higher concentration of Ca²⁺ for the enzyme activity [15].

We think that after p-Akt is released from cellular membranes and makes eNOS phosphorylated at Ser1177 and Ser (Thr) 613, p-Akt also increases [Ca²⁺]i, which enhances the activity of eNOS further. In this way, p-eNOS catalytes l-arginine to generate more NO (Figure 3).

To demonstrate the effect of ZX-5 on Cyclin D1 in HRECs, the expression of Cyclin D1 was assessed. Compared with DMSO-treated control cells, the protein expression level of Cyclin D1 was down-regulated in a concentration dependent manner in (R,R)ZX-5-treated cells, and (S,S)ZX-5 also down-regulated the expression of Cyclin D1 (Figure 5).

A CAM model was setup to evaluate the effect of (R,R)ZX-5 on angiogenesis. 48 h after drug administration into the chick embryos, medium and small blood vessels were counted within the 15 mm radius from the center of the drug carrier plate. The effect of ZX-5 on angiogenesis has been shown in Figure 6. Compared with negative control groups, the angiogenesis were inhibited by (R,R)ZX-5 (inhibition ratios were 34.24%, 44.00% and 65.68% at 7.5, 15 and 30 μg/mL, respectively) in a concentration dependent manner. (S,S)ZX-5 had no effect on the angiogenesis (inhibition ratio was 3.76% at 30 μg/mL) (Figure 7).

Angiogenesis is a complex process, usually including vascular basement membrane degradation, endothelial cell activation, proliferation, migration, re-formation of new blood vessels and vascular networks. Cyclin D1 is cell cycle-related protein interacting with multiple other proteins which acts on G1 phase and promotes cells to enter S phase. We tested the effect of ZX-5 on Cyclin D1 expression on protein level in HREC. It was found that (R,R)ZX-5 can significantly inhibit angiogenesis and down-regulate the expression of Cyclin D1. Downregulation of Cyclin D1 expression may partially contribute to (R,R)ZX-5’s anti-angiogenic effect. (S,S)ZX-5 also down-regulated Cyclin D1 expression, but it had no influence on angiogenesis.

The human retinal endothelial cells line (HRECs) was purchased from the American Type Culture (ATCC, Shanghai, China) and maintained in T75 flasks at 37 °C in an atmosphere of 5% CO₂. Cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, USA) supplemented with 10% fetal bovine serum (FBS), and antibiotics (penicillin G 100 U/mL, streptomycin sulfate; Generay Biotech Co., Ltd., Shanghai, China). The cells were allowed to reach confluence and then subcultured in 6-well plates at 8.0 × 10⁵ cells/well until confluent. To test the expression level of each protein, prepared cells were starved for 24 h in serum-free media before assay.

(R,R)ZX-5 and (S,S)ZX-5 were dissolved in Dimethyl sulfoxide (DMSO).

HRECs incubated in DMEM medium were divided into 3 groups: group 1 treated with DMSO in serum-free media, group 2 treated with (R,R)ZX-5 (7.5, 15, 30 μg/mL) in serum-free media, group 3 treated with (S,S)ZX-5 (7.5, 15, 30 μg/mL) in serum-free media. All cells were incubated for 24 h before western blot assay.

HRECs were seeded in 6-well culture plates with 8.0 × 10⁵ cells per well and incubated for 24 h. Then the cells were treated by (R,R)ZX-5 or (S,S)ZX-5 for 24 h. The cells were harvested with 0.05% trypsin/0.53 mM EDTA (Hyclone, UT, USA), washed with phosphate-buffered saline (PBS), and resuspended in 100 μL of all Mammalian protein Extraction Reagent (Generay Biotech Co., Ltd. Shanghai, China). Concentrated proteins were separated on a 12% gel for the detection of Cyclin D1, Akt, p-Akt (Thr473), a 8% gel for the detection of eNOS, p-eNOS (Ser1177), respectively. The separated proteins on the gel were transferred to PVDF membrane. Then the PVDF membrane (Millipore, MIT, USA) was incubated with a 1:1000 dilution of Cyclin D1, Akt, p-Akt (Thr473), eNOS, p-eNOS (Ser1177) and β-actin (Santa Cruz biotechnology, USA) antibodies. After incubation with the appropriate secondary antibodies (Multi Sciences Biotech Co., Ltd., Hang Zhou, China), blots were treated with the ECL reagents (Beyetime, Jiangsu province, China) and exposed to photographic film to detect protein expression.

Fluo-3/AM (Multi Sciences Biotech Co., Ltd., Hang Zhou, China) was used as an intracellular free Ca²⁺ fluorescent probe. In short, after the treatment with 30 μg/mL (R,R)ZX-5 or (S,S)ZX-5 respectively, cells were incubated with 3 μg Fluo-3/AM for 30 min at 37 °C (the final concentration of extracellular Fluo-3/AM was 3 μg/mL). Then the cells were washed 3 times with D-Hank’s solution to remove the extracellular Fluo-3/AM and the intracellular fluorescence was detected using a LSCM (Olympus Fv-1000, Olympus Corporation, Japan) with the excited light at a wavelength of 488 nm and the emission light at 525 nm.

The eggs which have been fertilized 6 days were used for the experiment and were incubated at 70% of humidity and 37 °C. A window was opened on the top of each egg after 1 day incubation. The windows were covered with sterile tape and the eggs were returned to the incubator. After another 1 day, the transparent tape was cut open and a drug carrier plate with a 15 mm diameter was placed in the chamber. Twenty microliters of each test reagent was added onto the plate. Test substances including (R,R)ZX-5 (the dose of ZX-5 was 7.5 μg/mL, 15 μg/mL, 30 μg/mL, respectively) and (S,S)ZX-5 (30 μg/mL). The physiological saline (0.1% DMSO) and endostatin (5 mg/mL) were used as negative and positive controls, respectively. Forty-eight hours after implantation of the filter paper disc, the pseudo-air chamber of chick embryos were opened and fixed in the fixative (formaldehyde-acetone, 1:1) for 5 min. The chick embryo was subsequently removed, and the CAM was carefully peeled off from the egg shell and laid out flat for photography. The number of medium-small blood vessels within a radius of 15 mm from the center of the carrier plate was counted.

Data were presented as the mean ± SD. The statistical significance of the differences was determined by the method of analysis of variance (ANOVA) or unpaired two-tailed t-tests; a value of p < 0.05 was considered significant.