Activating KRAS Mutations Expressed in 3D Endothelial Spheroids Induce Blebbing Morphologies Associated with...

Area of Science:

• Vascular Biology
• 3D Cell Culture Models
• Molecular Mechanisms of Disease

Background:

• Sporadic brain arteriovenous malformations (AVMs) are complex vascular malformations.
• Understanding the molecular drivers of AVM pathogenesis is crucial for developing effective treatments.

Purpose of the Study:

• To establish and characterize a 3D endothelial spheroid model expressing gain-of-function KRAS mutations.
• To investigate the molecular changes and cellular behaviors associated with KRAS mutations in the context of AVM development.

Main Methods:

• Human cerebral microvascular endothelial cells were cultured into 3D spheroids.
• Recombinant adeno-associated virus was used to express KRAS G12V in spheroids.
• Live-cell imaging was employed to monitor spheroid behavior over extended culture periods.

Main Results:

• KRAS G12V expression accelerated spheroid growth and sprouting, mimicking AVM characteristics.
• Novel phenotypes, including dynamic blebbing and amoeboid-like migration, were observed in KRAS G12V spheroids.
• MEK, mTOR, and Rho/ROCK inhibitors modulated spheroid growth and blebbing, indicating potential therapeutic pathways.

Conclusions:

• The 3D endothelial spheroid model effectively recapitulates key features of brain AVMs.
• This model reveals novel KRAS-driven endothelial cell behaviors relevant to AVM pathogenesis.
• The model provides a platform for further investigation of AVM driver mutations and therapeutic strategies.