Imidazole-based alkaloid derivative LCB54-0009 suppresses ocular angiogenesis and lymphangiogenesis in models of experimental retinopathy and corneal neovascularization

Abstract

Background and purpose: Abnormally induced angiogenesis and lymphangiogenesis are associated with human diseases, including neovascular eye disease. Substances that inhibit these processes may have potential as an attractive therapeutic strategy for these diseases.

Experimental approach: In vitro and in vivo angiogenesis and/or lymphangiogenesis were assessed in VEGF- or hypoxia-stimulated endothelial and retinal cells and in animal models of oxygen-induced retinopathy (OIR), streptozotocin-induced diabetic retinopathy (SIDR), suture-induced inflammatory corneal neovascularization (SICNV) and silver nitrate-induced corneal neovascularization. HUVECs and retinal cells were cultured under hypoxic conditions or incubated with VEGF to identify the molecular mechanisms involved.

Key results: The imidazole-based alkaloid derivative LCB54-0009 inhibited capillary-like tube formation in VEGF-induced HUVECs without inducing cytotoxic effects. Intravitreal injection of LCB54-0009 into retinas suppressed the formation of the pathological neovascular tufts and increased vascular permeability in both OIR of mice and SIDR of rats. Furthermore, subconjunctival injection of LCB54-0009 into the cornea suppressed corneal inflammation and inflammation-associated angiogenesis and lymphangiogenesis in SICNV of mice and silver nitrate cauterization of rats. These pharmacological activities were associated with effects on HIF-1α protein stability and HIF-1α/NF-κB redox sensitivity through its antioxidant activities. LCB54-0009 also inhibited the hypoxia-induced expression of angiopoietin-2, and VEGF-induced VEGFR-2 activation and downstream signalling, resulting in the down-regulation of the expression of pro-angiogenic factors and pro-inflammatory mediators and an up-regulation of the expression of anti-angiogenic factors.

Conclusions and implications: LCB54-0009 is a potential candidate molecule for blocking pathological angiogenesis and lymphangiogenesis mediated by HIF-1α- angiopoietin-2 expression and VEGFR-2 activation.

Figure 1

LCB54-0009 inhibits VEGF-induced angiogenesis in vitro. (A) Chemical structure of LCB54-0009 (C₂₄H₂₇N₃O₆, MW = 453.49, ≥97% purity). (B) Serum-starved HUVECs were seeded onto the Matrigel and incubated with vehicle (0.1% DMSO) or LCB54-0009 (20 μM) in the presence or absence of VEGF (20 ng·mL⁻¹) for 14 h. Capillary-like tube formation was viewed by using an inverted microscope. (C) HUVECs were incubated with vehicle (0.1% DMSO) or LCB54-0009 (20 μM) in the presence or absence of VEGF (20 ng·mL⁻¹) for 48 h. The mRNA levels were determined by quantitative real-time PCR. (D) HUVECs were incubated with various concentrations of LCB54-0009 for 24 to 72 h and cell viability was measured using the WST-1 reagent. Results are expressed as the mean ± SD of three independent experiments (n = 3). #P < 0.05 versus vehicle-treated group; *P < 0.05 and **P < 0.005 versus VEGF-stimulated group.

Figure 2

LCB54-0009 suppresses pathological angiogenesis and vascular leakage. (A–C) Intravitreal injection of vehicle (0.1% DMSO), LCB54-0009 (10 μg) or VEGF-trap (10 μg) in OIR mice (n = 5 per group). Vascular regression in the central region (A), pathological NVTs (B) and avascular area (C) were measured in the middle and peripheral regions of the retinas. Scale bars = 200 μm. (D–F) FITC-dextran was injected i.v. 1 h before the OIR mice were killed (D). Extravasated FITC-dextran (E) and extravasated Ter119⁺ cells (F) were measured within the retina near the NVT. Scale bars = 100 μm. *P < 0.05 and **P < 0.005 versus control mice. CTL, control; LCB54, LCB54-0009; V-trap, VEGF trap.

Figure 3

LCB54-0009 inhibits corneal neovascularization and inflammation-associated lymphangiogenesis. (A–E) Proliferation of blood vessels (BV) and lymphatic vessels (LV) were generated in the SICNV mouse model (n = 5 per group). Vehicle (0.1% DMSO), LCB54-0009 (100 μg) or VEGF trap (100 μg) was injected subconjunctivally on the day of corneal suture placement. Corneal angiogenesis, lymphangiogenesis and inflammation were determined by staining with CD31 (red), LYVE-1 (green) and CD11b (blue) antibodies respectively (A). BV diameter (B), BV density (C), LV density (D) and the infiltration area of inflammatory cells (E) were measured. Scale bars = 500 μm. (F and G) Corneal neovascularization was induced by the subconjunctival injection of silver nitrate to cauterize the cornea of rats (n = 5 per group). Vehicle (1% DMSO), LCB54-0009 (50 μg) or Avastin (200 μg) was injected subconjunctivally three days after silver nitrate cauterization and measured using an ophthalmic microscope with a digital camera. Original magnification was ×100. Avastin was used as a positive control. *P < 0.05 and **P < 0.005 versus control mice. CTL, control; LCB54, LCB54-0009; V-trap, VEGF trap.

Figure 4

LCB54-0009 inhibits hypoxia-induced increased levels of HIF-1α, angiogenic factors and inflammatory mediators. HUVECs were incubated with LCB54-0009 for 6 h in hypoxia. (A–C) HIF-1α levels were determined by Western blotting (A and B) or immunofluorescence (C). GAPDH served as a loading control. (D and E) The mRNA levels of angiogenic factors (D) and inflammatory mediators (E) were determined by quantitative real-time PCR. Results are expressed as the mean ± SD of three independent experiments (n = 3). #P < 0.05 versus vehicle-treated group; *P < 0.05 and **P < 0.005 versus hypoxia-induced group; ^†P < 0.05 and ^††P < 0.005 versus LCB54-0009-treated group.

Figure 5

LCB54-0009 regulates HIF-1α stability and HIF-1α/NF-κB redox sensitivity. (A and D) HUVECs were incubated for 6 h in hypoxia and the mRNA levels were determined by quantitative real-time PCR. Results are expressed as the mean ± SD of three independent experiments (n = 3). #P < 0.05 versus vehicle-treated group; *P < 0.05 and **P < 0.005 versus hypoxia-induced group; ^†P < 0.05 and ^††P < 0.005 versus LCB54-0009-treated group. (B and C) HUVECs were incubated with DFX (200 μM) in the presence or absence of LCB54-0009 (20 μM) in hypoxia for 6 h. Anti-VHL or anti-HIF-1α immunoprecipitates were analysed by Western blot analysis.

Figure 6

LCB54-0009 exhibits antioxidant activities. (A) The mixtures of hypoxia-stimulated HUVEC lysates, LCB54-0009 and Cu²⁺-containing working solution were incubated at room temperature for 1.5 h. Total antioxidant capacity (TAC) was measured as absorbance at 570 nm. (B) LCB54-0009 was mixed with DPPH solution and the mixtures were incubated at 25°C for 30 min. DPPH radical scavenging activity was measured as absorbance at 517 nm. (C) Superoxide radical was produced by the NADH/PMS system and the scavenging activity was measured as absorbance at 567 nm, followed by incubation at 25°C for 5 min. (D) HUVECs were incubated with LCB54-0009 in combination with stimuli (LPS 1 μg·mL⁻¹, TNF-α 10 ng·mL⁻¹, IL-1β 5 U·mL⁻¹ and IFN-γ 100 U·mL⁻¹) for 36 h. Amounts of NO were measured using a Griess reaction. Results are expressed as the mean ± SD of three independent experiments (n = 3). *P < 0.05 and **P < 0.005 versus control group (A–C); #P < 0.05 versus vehicle-treated group; *P < 0.05 and **P < 0.005 versus stimuli-induced group (D).

Figure 7

LCB54-0009 inhibits Ang-2 levels and VEGFR-2-signalling cascades. (A–C) HUVECs were incubated for 6 h in hypoxia or 48 h with VEGF (20 ng·mL⁻¹) in the presence or absence of LCB54-0009 (20 μM). The mRNA levels were determined by quantitative real-time PCR (A and B) or reverse transcription PCR (C). Results are expressed as the mean ± SD of three independent experiments (n = 3). #P < 0.05 versus vehicle-treated group; *P < 0.05 versus hypoxia- or VEGF-induced group; ^†P < 0.05 versus LCB54-0009-treated group. (D) HUVECs were incubated with LCB54-0009 for 1 h and then stimulated with VEGF (20 ng·mL⁻¹) for 15 min. Western blotting was performed from the cell lysates. GAPDH served as a loading control.

Figure 8

Proposed modes of action of LCB54-0009. LCB54-0009 inhibits ROS-mediated signalling cascades, resulting in the inhibition of HIF-1α, TLR/NF-κB and VEGF/VEGFR-2 signalling pathways. LCB54-0009 also inhibits VEGF-induced Ang-1/Tie-2 signalling cascades. LCB54, LCB54-0009.