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Three-dimensional Angiogenesis Assay System using Co-culture Spheroids Formed by Endothelial Colony Forming Cells and Mesenchymal Stem Cells
Published on: September 18, 2019
Bevacizumab and ranibizumab on microvascular endothelial cells: A comparative study
Raquel Costa1, Angela Carneiro, Ana Rocha
1Department of Biochemistry (U38-FCT), Faculty of Medicine, University of Porto, 4200-319 Porto, Portugal.
Abstract:
Given its broad effects in endothelium, vascular endothelial growth factor (VEGF) represents the primary rate-limiting step of angiogenesis. Therefore, VEGF targeting therapies were soon developed. Bevacizumab and ranibizumab are two of these therapeutic agents already in clinical use. Bevacizumab was first used for cancer treatment, whereas ranibizumab was designed to target choroidal neovascularization, the main cause of blindness in age-related macular degeneration. The present study aims to compare the multiple effects of bevacizumab and ranibizumab in human microvascular endothelial cells (HMECs). HMEC cultures were established and treated during 24 h with the anti-VEGF agents within the intravitreal-established concentration range or excipients. Analyses of VEGF content in cell media and VEGF receptor-2 (VEGFR-2) expression in cell lysates were performed. No cell cytotoxicity (MTS assay) was found in anti-VEGF-treated cultures at any concentration. Apoptosis (TUNEL assay) was significantly increased and cell proliferation (BrdU assay), migration (transwell assay) and assembly into vascular structures were significantly reduced by incubation with both agents at the two doses used. These findings were accompanied by a strong decrease in VEGF release, and in phosphorylated VEGFR-2 and Akt expression for both agents at the clinical concentration. Interestingly, phosphorylated Erk was only significantly reduced upon bevacizumab treatment. In addition, proliferation was more affected by ranibizumab, whereas migration, capillary formation, and phosphorylated VEGFR2 expression were significantly reduced by bevacizumab as compared to ranibizumab. Therefore, although both agents presented anti-angiogenic actions, distinct effects were exerted by the two molecules in HMEC. These findings suggest that a careful confirmation of these effects in clinical settings is mandatory.
Insights
Bevacizumab and ranibizumab, anti-vascular endothelial growth factor (VEGF) therapies, both inhibit angiogenesis in human microvascular endothelial cells (HMECs). However, they exhibit distinct effects on cell proliferation, migration, and signaling pathways.
Area of Science:
- Molecular Biology
- Cell Biology
- Pharmacology
Background:
- Vascular Endothelial Growth Factor (VEGF) is crucial for angiogenesis and is a target for therapies like bevacizumab and ranibizumab.
- Bevacizumab and ranibizumab are clinically used anti-VEGF agents, with distinct approved indications.
- Understanding their comparative effects on endothelial cells is vital for optimizing therapeutic strategies.
Purpose of the Study:
- To compare the in vitro effects of bevacizumab and ranibizumab on human microvascular endothelial cells (HMECs).
- To investigate the impact of these anti-VEGF agents on cellular processes and signaling pathways.
Main Methods:
- HMEC cultures were treated with bevacizumab or ranibizumab at clinically relevant concentrations.
- Assays included MTS for cytotoxicity, TUNEL for apoptosis, BrdU for proliferation, transwell for migration, and Western blot for protein expression (VEGF, VEGFR-2, p-VEGFR-2, Akt, p-Akt, Erk, p-Erk).
- VEGF levels in cell media were also analyzed.
Main Results:
- Neither agent exhibited cytotoxicity. Both increased apoptosis and reduced proliferation, migration, and vascular structure formation.
- Both decreased VEGF release, phosphorylated VEGFR-2, and phosphorylated Akt.
- Bevacizumab uniquely reduced phosphorylated Erk, while ranibizumab showed a greater impact on proliferation, and bevacizumab on migration and capillary formation.
Conclusions:
- Bevacizumab and ranibizumab demonstrate comparable anti-angiogenic actions in HMECs but with distinct molecular and cellular effects.
- Differences in effects on proliferation, migration, and specific signaling pathways warrant further clinical investigation.
- These findings highlight the need for careful consideration of agent-specific profiles in clinical applications.