The Chinese medicine formula HB01 reduces choroidal neovascularization by regulating the expression of vascular endothelial growth factor

Abstract

Background: Choroidal neovascularization (CNV) remains the leading cause of newly acquired blindness in the developed world. Currently anti-vascular endothelial growth factor (VEGF) therapies are broadly used to treat neovascular ocular disorders. Here we demonstrate the effect of a traditional Chinese medicine formula, HB01, on CNV.

Methods: A rat model of laser-induced CNV was used to investigate the effect of HB01 in vivo. The CNV lesions in the eye were evaluated using fundus fluorescein angiography and visualized/quantified using confocal microscopy. Expression of VEGF in the choroidal and retinal tissues was measured using quantitative real-time PCR and immunohistochemistry.

Results: We demonstrated that a traditional Chinese Medicine formula, named HB01, significantly reduced neovascularization in a rat CNV model. The effect of HB01 on CNV was comparable to the intravitreal injection of bevacizumab (Avastin). Our results also suggested that HB01 may reduce CNV partially through inhibiting the expression of VEGF.

Conclusions: These data support HB01 as an alternative therapy for ocular neovascular disorders.

Figure 1

Fundus fluorescein angiography. (A) Fundus fluorescein angiography of a representative rat in the Control group that did not received laser or drug treatment. (B) Fundus fluorescein angiography of a representative rat in the CNV group that received laser on Day 7 but not drug treatment. (C) Fundus fluorescein angiography of a representative rat in the CNV with bevacizumab group that received laser on Day 7 and one intravitreal injection of bevacizumab on Day 14. (D) Fundus fluorescein angiography of a representative rat in the CNV with HB01 group received laser on Day 7 and HB01 treatment for four weeks. Arrows marked lesions.

Figure 2

The frequency of CNV-positive spots. The CNV lesions were evaluated on Day 14, Day 21, and Day 28. The percentage of CNV positive spots was calculated as the ratio of CNV positive spots (in all eyes within a group of rats) and all CNV lesions.

Figure 3

Histologic analysis of CNV lesions in rat eyes. Paraffin-embedded cross sections through CNV lesions were examined using Hematoxylin/eosin (H&E) staining for eyes from the Control (A), CNV (B), CNV with bevacizumab (C), and CNV with HB01 (D) groups on Day 28. The thickness of the retinal lesion centers was summarized in (E). *P < 0.01, Mann Whitney test; NS, not significant. Arrows marked CNV lesions.

Figure 4

Confocal microscopy of CNV lesions. The fluorescent images of CNV in RPE-choroid-sclera flat mounts from the Control (A), CNV (B), CNV with bevacizumab (C), and CNV with HB01 (D) groups were shown. The vasculature was labeled with FITC-dextran via cardiac perfusion on Day 28. The total sizes of neovascular areas for eyes from CNV, CNV with bevacizumab, and CNV with HB01 were summarized in (E). *P < 0.01, Mann Whitney test; NS, not significant.

Figure 5

Expression ofVegfamRNA. Shown is the relative expression of Vefga mRNA on Day 28 in choroidal and retinal tissues (n = 12 for each group). Values are mean ± SD. *P < 0.01, Mann Whitney test; NS, not significant.

Figure 6

Expression of VEGF protein. Shown is the immunostaining of VEGF in the CNV lesions on Day 28 in eyes from the Control (A), CNV (B), CNV with bevacizumab (C), and CNV with HB01 (D) groups. The relative expression of VEGF protein was summarized in (E). *P < 0.01, Mann Whitney test; NS, not significant. Arrows marked CNV lesions.