Suppression of Pathological Ocular Neovascularization by a Small Molecular Multi-Targeting Kinase Inhibitor, DCZ19903

Abstract

Purpose: The administration of anti-vascular endothelial growth factor agents is the standard firs-line therapy for ocular vascular diseases, but some patients still have poor outcomes and drug resistance. This study investigated the role of DCZ19903, a small molecule multitarget kinase inhibitor, in ocular angiogenesis.

Methods: The toxicity of DCZ19903 was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assays, flow cytometry, Calcein-AM/PI staining, and terminal uridine nick-end labeling staining. Oxygen-induced retinopathy and laser-induced choroidal neovascularization models were adopted to assess the antiangiogenic effects of DCZ19903 by Isolectin B4 (GS-IB4) and hematoxylin-eosin staining. EdU assays, transwell migration assays, tube formation, and choroid sprouting assays were performed to determine the antiangiogenic effects of DCZ19903. The antiangiogenic mechanism of DCZ19903 was determined using network pharmacology approach and western blots.

Results: There was no obvious cytotoxicity or tissue toxicity after DCZ19903 treatment. DCZ19903 exerted the antiangiogenic effects in OIR model and choroidal neovascularization model. DCZ19903 inhibited the proliferation, tube formation, migration ability of endothelial cells, and choroidal explant sprouting. DCZ19903 plus ranibizumab achieved greater antiangiogenetic effects than DCZ19903 or ranibizumab alone. DCZ19903 exerted its antiangiogenic effects via affecting the activation of ERK1/2 and p38 signaling.

Conclusions: DCZ19903 is a promising drug for antiangiogenic treatment in ocular vascular diseases.

Translational relevance: These findings suggest that DCZ19903 possesses great antiangiogenic potential for treating ocular vascular diseases.

Figure 1.

Synthesis of DCZ19903.

Figure 2.

DCZ19903 administration has no obvious cytotoxicity and tissue toxicity. (A–C) HUVECs were treated with DCZ19903 (1 nM to 100 µΜ), or left untreated (Ctrl) for 24 hours. MTT assays were used to evaluate cell viability (A; n = 4). (B) Annexin V-FITC/PI assays were used to quantify the apoptotic percentage of HUVECs (B, n = 4). Calcein-AM/PI staining was used to detect cell apoptosis (C) (n = 4, scale bar, 20 µm). ^*P < 0.05 versus Ctrl group. (D and E) C57BL/6J mice received intravitreal injections of PBS (Ctrl), DMSO, or DCZ19903 (1 µg/µL). At day 7 after the injection, the histological changes and cell apoptosis in the retinas were evaluated by HE staining and TUNEL staining assays. In TUNEL staining experiment, DNase I was detected as the positive control (n = 4, scale bar: 50 µm).

Figure 3.

DCZ19903 administration inhibits ocular angiogenesis in vivo. (A and B) Laser-induced CNV models were used to determine the antiangiogenic effects of DCZ19903. After the laser injury, the mice received intravitreal injections of 10% DMSO (Ctrl), DCZ19903 (1 µg/µL), ranibizumab (10 mg/mL), or DCZ19903 (1 µg/µL) plus ranibizumab (10 mg/mL), respectively. HE staining was used to measure the area of neovascular lesions after 7 days. The thickness was calculated from the bottom of the choroid to the top of the lesion, as indicated by the yellow line. The area of the lesion was measured by Image J (A) (n = 4, scale bar, 50 µm). CNV formation was observed by GS-IB4 staining (B) (n = 4, scale bar, 100 µm). (C) P7 mouse pups were exposed to hyperoxia (75% O₂) for 5 days with their nursing mothers. Subsequently, they were returned to normoxic condition and injected intravitreally with 10% DMSO (Ctrl), DCZ19903 (1 µg/µL), ranibizumab (10 mg/mL), or DCZ19903 (1 µg/µL) plus ranibizumab (10 mg/mL) at P12. To evaluate retinal vasculature, the retinas were extracted at P17 and stained with GS-IB4. Avascular regions were highlighted by white dashed lines. Angiogenic regions were highlighted by yellow markers (n = 4) (scale bar, 200 µm). ^*P < 0.05 versus Ctrl group. ^△P < 0.05 versus DCZ19903 + Ran group.

Figure 4.

DCZ19903 inhibits endothelial angiogenic function in vitro. (A–D) HUVECs were incubated with VEGF (10 ng/mL), VEGF (10 ng/mL) plus DCZ19903 (50 nM), VEGF (10 ng/mL) plus ranibizumab (100 µg/mL), VEGF (10 ng/mL) plus DCZ19903 (50 nM) and ranibizumab (100 µg/mL), or left untreated (Ctrl). Cell viability was detected by MTT assays (A, n = 4). Cell proliferation was measured by EdU assays (B) (n = 4; scale bar, 20 µm). Cell migration was detected by transwell assays (C) (n = 4; scale bar, 20 µm). Tube formation was observed under a light microscope (D) (n = 4; scale bar, 100 µm). (E) The RPE/choroid complexes of C57BL/6J mice were prepared and sliced into 1 mm × 1 mm pieces and then placed in 24-well plates precoated with Matrigel. The sprouting potency of choroidal explants were observed on day 4, day 5, and day 6 after seeding. Quantification of sprouting area and representative images of choroidal sprouting were shown (E) (n = 4; scale bar, 200 µm). ^* P < 0.05 versus Ctrl group; ^#P < 0.05 versus VEGF group; ^△P < 0.05 DCZ19903 or Ran versus DCZ19903 + Ran group.

Figure 5.

DCZ19903 inhibits vascular permeability. (A and B) HUVECs were incubated with VEGF (10 ng/mL), VEGF plus DCZ19903 (50 nM), VEGF plus ranibizumab (100 µg/mL), VEGF plus DCZ19903 (50 nM) and ranibizumab (100 µg/mL), or left untreated (Ctrl). qRT-PCR assays were performed to assess the effects of DCZ19903 administration on ICAM-1 expression (A, n = 4). Evans Blue-transwell experiments were used to detect the role of DCZ19903 treatment on the permeability of HUVECs induced by VEGF (B) (n = 4). (C) P7 pups were exposed to hyperoxia (75% O₂) for 5 days with their nursing mothers. Subsequently, they were returned to normoxic condition and injected intravitreally with 10% DMSO (Ctrl), DCZ19903 (1 µg/µL), ranibizumab (10 mg/mL), or DCZ19903 (1 µg/µL) plus ranibizumab (10 mg/mL) at P12. WT indicates the wild-type group without treatment. Immunofluorescence analysis of ICAM-1 was performed at P17 to detect the expression of ICAM-1 in OIR retinas. Representative images and quantitative data were presented (n = 4; scale bar, 50 µm; nuclei, blue; ICAM-1–positive cells, green). (D and E) Laser-induced CNV models were used to determine the antiangiogenic effects of DCZ19903. After laser injury, the mice received intravitreal injections of 10% DMSO (Ctrl), DCZ19903 (1 µg/µL), ranibizumab (10 mg/mL), or DCZ19903 (1 µg/µL) plus ranibizumab (10 mg/mL). The wild-type mice received no treatment (WT). qRT-PCRs and western blots revealed that DCZ19903 administration reduced the expression of ICAM-1 in CNV model. GAPDH was detected as the internal control (n = 4). ^*P < 0.05 versus Ctrl group; ^#P < 0.05 versus VEGF group; ^△P < 0.05 versus DCZ19903 + Ran group.

Figure 6.

DCZ19903 exerts antiangiogenic effects via regulating MAPK signaling. (A) Network diagram of interaction between DCZ19903 and ocular neovascularization. (B) PPI network analysis. (C) GO enrichment analysis. (D) KEGG pathway enrichment analysis. (E) HUVECs were treated with DCZ19903, ranibizumab, or DCZ19903 plus ranibizumab for 24 hours before being stimulated with VEGF (50 ng/mL). The proteins were electrophoresed, transferred to the membranes, and probed with the specified antibodies. (F) HUVECs were pretreated with or without SB203580, U0126, and then cultured with VEGF (50 ng/mL), VEGF plus DCZ19903 (50 nM), or left untreated (Ctrl). GAPDH was detected as the internal control for protein loading (n = 4). ^*P < 0.05 versus VEGF group; ^△P < 0.05 versus DCZ19903 + Ran group.