Difference between revisions of "Part:BBa K5374018"

Latest revision as of 11:25, 23 September 2024

CBD MMPs-VEGF (Collagen-Binding Domain from Matrix Metalloproteinases fused with Vascular Endothelia

The CBD from MMPs binds strongly to collagen, ensuring that the fusion protein localizes to collagen-rich environments. VEGF (Vascular Endothelial Growth Factor) is a key signaling protein that promotes blood vessel formation, critical for tissue repair and regeneration. By fusing VEGF with the collagen-binding domain, the protein can be directed specifically to collagen scaffolds, allowing for localized and sustained angiogenesis in tissue engineering applications.

For this part, CBD MMPs-VEGF, expression in E. coli BL21 (DE3) yielded high concentrations of soluble protein under standard conditions, confirming that this fusion protein could be expressed easily and efficiently.

We characterized the sustained-release activity and biological activity of this part.

1 Sequential release

In this experiment, the release profiles of CBD MMPs-VEGF and FTD-BMP-4 from collagen hydrogels were monitored over time. The two fusion proteins were incorporated into collagen hydrogels, and their release was quantified using ELISA assays. The hydrogels were incubated in PBS at 37°C, with supernatant samples collected at regular intervals to measure the amount of protein released. After each sampling, fresh PBS was added to the hydrogels to maintain consistent conditions throughout the experiment.

Fig1.1 Time-Dependent Release Profiles of CBD MMPs-VEGF and FTD-BMP-4 from Collagen Hydrogel

The results reveal a significant difference in the release kinetics between the two cell factors. CBD MMPs-VEGF showed a rapid and early release pattern, starting with an accelerated increase in the release rate during the first 10 days. By day 10, more than 50% of the VEGF had already been released from the hydrogel. This sharp increase continued, reaching approximately 75% release by day 20. Following this, the release rate gradually slowed, with the curve plateauing as nearly 100% of the VEGF was released by day 30. The release curve of VEGF shows an initial rapid release phase, followed by a stabilization period where the release rate gradually levels off.This fast release behavior can be attributed to the lower binding affinity of the CBD MMPs domain, allowing VEGF to dissociate from the collagen matrix more quickly.

2 In vitro biological activity(VEGF)

The tube formation assay is a widely used in vitro method for assessing the ability of endothelial cells, such as HUVECs (Human Umbilical Vein Endothelial Cells), to form capillary-like structures, mimicking the process of angiogenesis. This assay is particularly useful for evaluating the angiogenic potential of cell factors, small molecules, or other treatments.

In the assay, endothelial cells are seeded on a Matrigel matrix, which provides an extracellular matrix environment that encourages the cells to migrate, align, and connect with each other, forming tubular networks. When angiogenic cell factors, such as VEGF, are present, these processes are enhanced, leading to more extensive and faster formation of tube-like structures.

The process of cell connection and tube formation involves several key cellular mechanisms:

1. Migration: Endothelial cells move toward each other, driven by chemotactic signals, such as VEGF, which promote cell motility. VEGF binds to its receptors on endothelial cells, activating intracellular signaling pathways that guide this migration.
2. Adhesion: As cells come into contact, they adhere to each other via cell-cell adhesion molecules such as VE-cadherin. These molecules facilitate stable connections between neighboring cells, which are critical for forming continuous structures.
3. Cell Structure Rearrangement: The internal framework of endothelial cells undergoes dynamic changes, allowing the cells to elongate and align with each other. This is crucial for shaping the cells into tube-like formations.
4. Lumen Formation: Once the cells are aligned and connected, they start forming hollow tubes that resemble capillaries. This involves the coordinated action of intracellular vacuoles and the fusion of these vacuoles between neighboring cells.

Overall, the tube formation assay provides insights into the angiogenic potential of treatments like CBD MMPs-VEGF, as seen in our experiment. The results suggest that CBD MMPs-VEGF promotes cell migration, adhesion, and network formation, confirming its ability to stimulate angiogenesis through effective cell factor release.

In the tube formation assay, human umbilical vein endothelial cells (HUVECs) were seeded onto a Matrigel-coated plate to facilitate tube formation. The experimental group was treated with CBD MMPs-VEGF at a final concentration of 100 ng/ml, while the control group received no VEGF. The cells were incubated at 37°C with 5% CO₂ for 4-6hours, during which tube formation was observed. The formation of capillary-like structures was monitored under a microscope to assess angiogenesis. The extent of tube formation between the experimental and control groups was compared to determine the impact of CBD MMPs-VEGF on promoting angiogenesis.

Fig1.2 Comparison of Tube Formation in HUVECs a) Control; b)CBD MMPs-VEGF Treatment

Image a (Without CBD MMPs-VEGF): In the control group, where no CBD MMPs-VEGF was added, the HUVECs exhibited weak angiogenic activity. Only a few scattered cell connections were observed, with minimal evidence of network formation. Quantitatively, the tube formation rate (measured by parameters such as total tube length, number of branch points, and closed loops or tube closure rate) was significantly reduced. The tube closure rate, which measures the percentage of formed capillary-like loops, was close to zero, reflecting the lack of organized tube structures. This result confirms that without the presence of VEGF, the cells are unable to initiate or sustain effective angiogenesis.

Image b (CBD MMPs-VEGF at 100 ng/ml): In the experimental group treated with 100 ng/ml CBD MMPs-VEGF, the HUVECs showed robust tube formation, with clearly defined and well-organized capillary-like structures. Quantitative analysis revealed a dramatic increase in several angiogenesis indicators. The tube closure rate increased to approximately 70-80%, indicating a high number of complete tube loops. Additionally, the total tube length and number of branch points were significantly higher compared to the control, demonstrating enhanced network complexity and cell connectivity. These findings suggest that the addition of VEGF significantly promoted angiogenesis, with the cells forming a dense and interconnected tubular network, characteristic of active capillary formation.

This comparison highlights the essential role of CBD MMPs-VEGF in promoting angiogenesis, as quantified by standard tube formation metrics. The increased tube closure rate, total tube length, and branch points in the experimental group strongly indicate that the VEGF produced in the system retains its biological function and effectively stimulates endothelial cell organization and network formation.The comparison between these two images confirms that the CBD MMPs-VEGF produced in this study retains biological activity and effectively stimulates tube formation, validating its functional ability to promote angiogenesis.

Sequence and Features