Endothelial matrix assembly during capillary morphogenesis: insights from chimeric TagRFP-fibronectin matrix

Area of Science:

• Biomaterials Science
• Cell Biology
• Vascular Biology

Background:

• Three-dimensional extracellular matrix (ECM) is crucial for in vitro vasculogenesis.
• Existing models face challenges with fibroblast senescence and distinguishing cell-derived matrix.
• Immortalized fibroblasts offer a potential solution for stable matrix production.

Purpose of the Study:

• To develop and characterize a novel 3D extracellular matrix model for studying vasculogenesis.
• To utilize fluorescently tagged fibronectin to track matrix assembly and remodeling.
• To investigate endothelial cell interactions with the engineered matrix.

Main Methods:

• Utilized hTERT-immortalized WI-38 fibroblasts to create 3D matrices.
• Incorporated TagRFP-fibronectin into the matrix for visualization.
• Co-cultured matrices with Human Umbilical Vein Endothelial Cells (HUVEC).
• Analyzed ECM composition and HUVEC-driven matrix remodeling using microscopy.

Main Results:

• hTERT-immortalized WI-38 fibroblast matrices mimicked wild-type matrices in composition (tenascin-C, fibronectin, collagen VI, I, IV).
• TagRFP-fibronectin was successfully incorporated and localized within the 3D matrix.
• HUVECs formed 3D adhesions and tubes on the engineered matrices.
• The model allowed visualization of membrane type 1 metalloprotease and new HUVEC-derived matrix components.

Conclusions:

• hTERT-immortalized WI-38 fibroblast matrices serve as a robust platform for vasculogenesis research.
• The TagRFP-fibronectin labeling enables detailed observation of matrix assembly and remodeling.
• This model facilitates the study of dynamic events during endothelial cell tubulogenesis.