Post-surgical scarring is a known cause of trabeculectomy failure. The aim of this study was to investigate the effectiveness of ranibizumab as an adjuvant anti-scarring agent in experimental trabeculectomy. Forty New Zealand white rabbits were randomised into four eye treatment groups: groups A (control), B (ranibizumab 0.5 mg/mL), C (mitomycin C [MMC] 0.4 mg/mL), and D (ranibizumab 0.5 mg/mL and MMC 0.4 mg/mL). Modified trabeculectomy was performed. Clinical parameters were assessed on post-operative days 1, 2, 3, 7, 14, and 21. Twenty rabbits were euthanised on day 7, and the other twenty were euthanised on day 21. Eye tissue samples were obtained from the rabbits and stained with haematoxylin and eosin (H&E). All treatment groups showed a significant difference in IOP reduction compared with group A (p < 0.05). Groups C and D showed a significant difference in bleb status on days 7 (p = 0.001) and 21 (p = 0.002) relative to group A. H&E staining showed significantly low fibrotic activity (p < 0.001) in group C on both days and inflammatory cell grade in group B on day 7 (p < 0.001). The grade for new vessel formation was significantly low in groups B and D on day 7 (p < 0.001) and in group D on day 21 (p = 0.007). Ranibizumab plays a role in reducing scarring, and a single application of the ranibizumab-MMC combination showed a moderate wound-modulating effect in the early post-operative phase.
The anti-fibrotic properties of ranibizumab have been well documented. As an antagonist to vascular endothelial growth factor (VEGF), ranibizumab works by binding and neutralizing all active VEGF-A, thus limiting progressive cell growth and proliferation. Ranibizumab application in ocular diseases has shown remarkable desired effects; however, to date, its antifibrotic mechanism is not well understood. In this study, we identified metabolic changes in ranibizumab-treated human Tenon's fibroblasts (HTFs). Cultured HTFs were treated for 48 h with 0.5 mg/mL of ranibizumab and 0.5 mg/mL control IgG antibody which serves as a negative control. Samples from each group were injected into Agilent 6520 Q-TOF liquid chromatography/mass spectrometer (LC/MS) system to establish the metabolite expression in both ranibizumab treated cells and control group. Data obtained was analyzed using Agilent Mass Hunter Qualitative Analysis software to identify the most regulated metabolite following ranibizumab treatment. At p-value < 0.01 with the cut off value of two-fold change, 31 identified metabolites were found to be significantly upregulated in ranibizumab-treated group, with six of the mostly upregulated having insignificant role in fibroblast cell cycle and wound healing regulations. Meanwhile, 121 identified metabolites that were downregulated, and seven of the mostly downregulated are significantly involved in cell cycle and proliferation. Our findings suggest that ranibizumab abrogates the tissue scarring and wound healing process by regulating the expression of metabolites associated with fibrotic activity. In particular, we found that vitamin Bs are important in maintaining normal folate cycle, nucleotide synthesis, and homocysteine and spermidine metabolism. This study provides an insight into ranibizumab's mechanism of action in HTFs from the perspective of metabolomics.
Inhibiting exaggerated wound healing responses, which are primarily mediated by human Tenon's fibroblast (HTF) migration and proliferation, has become the major determining factor for a successful trabeculectomy. Antivascular endothelial growth factor (anti-VEGF) has showed promising results as a potential antifibrotic candidate for use concurrently in trabeculectomy. Preliminary cohort studies have revealed improved bleb morphology following trabeculectomy augmented with ranibizumab. However, the effects on HTFs remain unclear. This study was conducted to understand the effects of ranibizumab on transforming growth factor (TGF)-β1 and transforming growth factor (TGF)-β2 expression by HTFs.