Exploring Endothelial-Matrix Dynamics: Organization, Viscoelasticity, and Remodeling

Abstract

The vascular system relies on tightly regulated interactions between endothelial cells and their surrounding extracellular matrix (ECM) to maintain tissue homeostasis and support angiogenesis. Alterations in tissue mechanics are a hallmark of vascular dysfunction in aging and disease, yet establishing causal relationships between dynamic mechanical cues, endothelial behavior, and pathological outcomes remains challenging. In particular, the time-dependent and spatially heterogeneous mechanical properties of native tissues are poorly captured by conventional two-dimensional culture systems and static in vitro models. This dissertation addresses this gap by developing and applying experimental platforms to interrogate how dynamic tissue mechanics and endothelial cell–matrix interactions form feedback loops that drive vascular dysfunction.To establish causality between mechanical cues and endothelial responses, three complementary approaches were employed. First, three-dimensional in vitro endothelial culture platforms were developed to enable temporally and spatially controlled mechanical perturbations within collagen-based hydrogels, allowing precise modulation of local shear and stiffness without altering bulk material composition. Second, oscillatory atomic force microscopy (AFM)–based microrheology was characterized and applied to quantify both elastic and viscous tissue properties with high spatial resolution. This methodology was used to compare healthy and pathological vascular tissues, revealing distinct stiffness and viscoelastic signatures between healthy mouse retina and oxygen-induced retinopathy, and demonstrating the sensitivity of microrheology to disease-associated mechanical remodeling. Third, dynamic matrix stiffening was imposed in situ around endothelial cells and endothelial colony-forming cells to examine how evolving mechanical environments regulate endothelial matrix remodeling. Using metabolic labeling of nascent proteins, combined with local mechanical measurements, endothelial-driven ECM deposition and reorganization were quantified in response to stiffening. These studies demonstrate that endothelial cells actively remodel their local mechanical microenvironment in compliant matrices, establishing a reciprocal feedback loop between cell-generated forces and ECM mechanics. In contrast, dynamic matrix stiffening suppresses collagen reorganization and nascent protein deposition, despite unchanged bulk matrix density. Transcriptomic analysis revealed that stiffening reprograms endothelial progenitors from a matrix-constructive, angiogenic state toward a stress-responsive metabolic phenotype. Mechanistically, integrin α2 was identified as a critical mechanotransducer mediating stiffness-induced suppression of endothelial matrix remodeling, with inhibition of α2-integrin attenuating nascent protein synthesis and reducing adhesion strength. Together, this work establishes dynamic tissue mechanics as both a regulator and consequence of endothelial behavior and identifies mechanosensitive checkpoints that may underlie impaired vascular regeneration in fibrotic, aged, and diseased tissues. By integrating advanced micromechanical measurements with engineered in vitro models, this dissertation provides new mechanistic insight into how endothelial–ECM feedback loops contribute to vascular dysfunction and offers broadly applicable tools for studying mechanobiology in complex tissues.