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.
To date, the molecular signalling mechanisms which regulate growth factors-induced MSCs tenogenic differentiation remain largely unknown. Therefore, a study to determine the global gene expression profile of tenogenic differentiation in human bone marrow stromal cells (hMSCs) using growth differentiation factor 5 (GDF5) was conducted. Microarray analyses were conducted on hMSCs cultures supplemented with 100 ng/ml of GDF5 and compared to undifferentiated hMSCs and adult tenocytes. Results of QuantiGene® Plex assay support the use and interpretation of the inferred gene expression profiles and pathways information. From the 27,216 genes assessed, 873 genes (3.21% of the overall human transcriptome) were significantly altered during the tenogenic differentiation process (corrected p<0.05). The genes identified as potentially associated with tenogenic differentiation were ARHGAP29, CCL2, integrin alpha 8 and neurofilament medium polypeptides. These genes, were mainly associated with cytoskeleton reorganization (stress fibers formation) signaling. Pathway analysis demonstrated the potential molecular pathways involved in tenogenic differentiation were: cytoskeleton reorganization related i.e. keratin filament signaling and activin A signaling; cell adhesion related i.e. chemokine and adhesion signaling; and extracellular matrix related i.e. arachidonic acid production signaling. Further investigation using atomic force microscopy and confocal laser scanning microscopy demonstrated apparent cytoskeleton reorganization in GDF5-induced hMSCs suggesting that cytoskeleton reorganization signaling is an important event involved in tenogenic differentiation. Besides, a reduced nucleostemin expression observed suggested a lower cell proliferation rate in hMSCs undergoing tenogenic differentiation. Understanding and elucidating the tenogenic differentiation signalling pathways are important for future optimization of tenogenic hMSCs for functional tendon cell-based therapy and tissue engineering.