Date of Award

Spring 6-12-2026

Document Type

Dissertation

Degree Name

Ph.D. in Biological Sciences

Organizational Unit

College of Natural Science and Mathematics, Biological Sciences

First Advisor

Erich J. Kushner

Second Advisor

Dinah Loerke

Third Advisor

Schuyler B. van Engelenburg

Fourth Advisor

J. T. Blankenship

Copyright Statement / License for Reuse

All Rights Reserved
All Rights Reserved.

Keywords

Caveolae, Caveolin-1, Endothelial cells, Micropatterning, Spatial cell mapping

Abstract

Caveolae are specialized, flask-shaped membrane invaginations highly expressed in endothelium and dysregulated in atherosclerosis. Caveolae play a central role in buffering membrane tension, yet the principles governing their spatial organization remain elusive. Thus, we sought to generate the most comprehensive and systematic analysis of blood vessel caveolar spatial organization. However, cell culturing, the backbone of human-focused biological research, does not standardly do well to model physiologically relevant cell behaviors such as migration or polarity-based tissue formation, particularly for punctate proteins and structures like caveolae. Micropatterns are cell-adhesive shapes that biophysically confine cell(s) to a user defined geometry which stereotype organelle and cytoskeletal systems to better study morphologically relevant conditions. A major drawback of this approach is the cost of equipment and reagents associated with fabrication. Here, we provide a characterization of a compound called Lipidure (2-Methacryloyloxy ethyl phosphorylcholine) that is up to 40X less expensive than other cell repulsive coating agents. We demonstrated that Lipidure is sensitive to deep UV degradation for photolithography masking, non-toxic in prolonged culture, cell-repulsive, and effective at constraining cell geometry for quantification of cytoskeletal systems. In turn, we leveraged Lipidure-based micropatterning to impose precise biophysical constraints on endothelial cell geometry to study the organization of caveolae, probing defined tensional and polarized environments. We integrated a computational, high-throughput spatial cell mapping pipeline for analyzing thousands of caveolae, providing extremely high-fidelity analysis. Our results provide a governing framework of how total cellular caveolae are spatially organized during random and directional migration, non-motile polarized, nascent and stable monolayers with differing confinement levels as well as in angiogenic vasculature in vivo. Broadly, our results demonstrated caveolae preferentially organized in the rear of migrating and polarized endothelial cells. In differing monolayer configurations, caveolae default to a peri-junctional spatial organization. Lastly, in mouse retinal blood vessels caveolae are most prominent in the vascular front due to their responsiveness to vascular endothelial growth factor signaling. Overall, these results strongly suggest that caveolae cellular arrangement and number are highly predictive of vascular stability and remodeling states.

Copyright Date

6-2026

Publication Statement

Copyright is held by the author. User is responsible for all copyright compliance.

Rights Holder

Andrew B. Grespin

Provenance

Received from ProQuest

File Format

application/pdf

Language

English (eng)

Extent

92 pgs

File Size

2 MB

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