Abstract
Background
- High-molecular weight heparin (HMWH), a molecule which is extensively in use as an anticoagulant shows concentration-dependent angiogenic and anti-angiogenic potential. Based on the concentration, HMWH can bind with both angiogenic and anti-angiogenic factors and exerts diverse effect. Our earlier data suggested that HMWH (15 kDa) can induce concentration-dependent neovascularization on chicken chorioallantoic membrane (CAM). The diffusion pattern of HMWH through various layers of CAM supports its internalized action with the various cellular components of angiogenesis. So far, no studies have reported the interactive potential of HMWH with various pro-angiogenic growth factors under physiological conditions. Hence, we aimed to find the transcription level interaction of HMWH with major pro-angiogenic growth factors. In connection to the research, for the first time, we validated the three-dimensional structures of chicken-specific pro-angiogenic growth factors such as FGF2, MMP2, MMP9, NOS3, VEGF A, and VEGF C to find the binding affinity of HMWH with the core-functional units of these growth factors.
Methods
- CAMs are incubated with 50, 100, and 150 µM concentration of HMWH. Changes in the transcription level of specified pro-angiogenic growth factors are analyzed by semi-PCR method. The functional aspects of these molecules are identified with zymogram and immunohistochemical approaches. Scanning electron microscopic technique is applied to find the morphological changes on CAM under HMWH incubation. Three-dimensional structure validation and molecular docking are performed using the SWISS-MODEL web server and AutoDock vina-PyRx software version 8.0.
Results
- HMWH can enhance the transcription level of major pro-angiogenic growth factors with a significant impact on FGF2 and MMP2 under 100 µM concentration. The in-silico analysis reveals that HMWH shows a higher binding affinity with FGF2 followed by MMP2, MMP9, NOS3, VEGF A, and VEGF C, respectively.
Conclusion
- The combined results from the experimental and in-silico analysis reveal that HMWH can interact with pro-angiogenic growth factors under micromolar concentration in physiological conditions while inducing angiogenesis. This observation further supports the therapeutic benefits of HMWH as an angiogenic factor under micromolar concentration.