Investigating Transluminal Endothelial Bridging in a Microvessel-on-a-Chip

Angiogenesis is the process of new blood vessel generation that is essential to form and sustain new tissue in development and disease. Intussusceptive angiogenesis (IA), whereby blood vessels split into two, is one of the two main mechanisms of angiogenesis yet it remains poorly understood more than 35 years after its discovery. The hallmark of IA is the peculiar occurrence of endothelial cells that bridge the lumen and subsequently remodel with adjacent extracellular matrix to form a tissue pillar. How lining endothelial cells form transluminal bridges against the force of blood flow remains enigmatic, largely due to the challenges of visualising this seemingly stochastic event in vivo and the lack of in vitro models. To elucidate this bridging process, I employed a vessel-on-a-chip strategy where various transluminal endothelial bridge morphologies were revealed, ranging from filamentous strands to mature multicellular pillars with cores containing extracellular matrix. These bridge architectures recapitulate those in situ as determined by systematic literature review. Furthermore, I identified transluminal endothelial bridges in the dermis of patients with limb-threatening ischemia, with significantly higher density near the edge of non-healing wounds. Next, I undertook multipoint 3D timelapse microscopy of the microphysiological system which revealed the evolution of circumferentially orientated endothelial cell protrusions into transluminal bridges forming by partial delamination. I found that enhancing actomyosin tension by hyperactivation of Rho constrained bridging, whereas weakening traction forces by inhibition of non-muscle myosin II or weakening selective adhesion forces by blocking α5ß1 integrin enhanced bridging. These findings reveal that endothelial cells can relocate transluminally via controlled delamination from a curved substrate. Finally, I investigated the biochemical and biomechanical regulation of transluminal endothelial bridging. I found that, in contrast to endothelial sprouting, endothelial bridging was enhanced by the inhibition of VEGFR2 signalling and impairment of NO synthesis. Additionally, though bridges formed within a range of FSS from 0.02 dyn/cm² to 20 dyn/cm², complex multicellular bridges were exclusive to low shear stress environments. In summary, this thesis provides novel insights into the cell migration and adhesion dynamics involved in endothelial cell intraluminal translocation, as well as the biochemical and biomechanical regulation of this process. These findings have relevance to strategies for therapeutic modulation of IA in a variety of pathologic contexts.

Name thesis:degree_name

Ph D

Discipline thesis:degree_discipline

Medical Biophysics

Grantor dc:publisher

The University of Western Ontario

Year dc:date.issued

2025

Author dc:creator

• Staples, Sabrina

Advisor dc:contributor.advisor

• Pickering, J. Geoffrey

• angiogenesis
• intussusceptive angiogenesis
• endothelial cell
• microfluidics
• 3D cell culture
• 4D microscopy
• skin wounds
• cytoskeleton
• integrins
• VEGFR2
• confocal microscopy

Statement dc:rights

• Attribution-NonCommercial-NoDerivatives 4.0 International

Language dc:language.iso

en

Handle dc:identifier.uri

https://hdl.handle.net/20.500.14721/38550

OAI identifier oai:identifier

oai:uwo.scholaris.ca:20.500.14721/38550