PhD Dissertation Defense
Monday, April 14, 2025
50 Prescott Street, Room 4911
2:00pm-3:00 pm
"Investigating The Effects of Fibrotic Stiffening on Lymphatic Endothelial Cell Growth and Barrier Integrity Using Photocrosslinked collagen Matrices"
Brian Ruliffson
Abstract: Fibrosis is an integral part of many chronic diseases including kidney disease, cancerous tumors, and lymphedema. Lymphangiogenesis—new lymphatic capillary growth—can be triggered by fibrosis-related tissue stiffening and soluble factor signaling that occurs under disease conditions. New lymphatic vessel density can be used as a predictor of the severity of fibrotic progression, but despite the consistency of lymphatic capillary growth during fibrosis, tumors and kidney disease are treated with anti-lymphangiogenic therapies while lymphedema therapies promote lymphangiogenesis. The differences in these therapeutic approaches likely arise, in part, from a conflation of lymphangiogenesis (growth) and lymphatic capillary function (barrier integrity). Current preclinical in vitro models are inadequate when separating the two processes and do not match outcomes observed in vivo. Moreover, less is known about how tissue stiffening generally impacts lymphatic capillary growth and function, which is important when studying fibrosis. Current in vitro models have not yet included physiologically relevant stiffnesses to represent disease states, often opting to use soft hydrogel extracellular matrices (ECM) that are more representative of tissue stiffness during developmental stages. Studies also tend to focus on lymphatic vessel growth without fully considering and assessing function (e.g., barrier integrity) as a distinct outcome. Moreover, while some information is known about how ECM stiffening regulates lymphatic vessel growth via mechanosensitive molecules and growth factor receptor expression, less is known for whether those pathways play a role in stiffness-mediated changes in function. Therefore, improved in vitro models with controlled biophysical properties are needed to systematically investigate stiffness mediated outcomes and the distinct contributions to lymphatic capillary growth and function.
The work presented in this dissertation describes the use of methacrylated type I collagen (PhotoCol®, Advanced BioMatrix) as a culture substrate that can be stiffened with photo-crosslinking as an investigative tool to study lymphatic endothelial cell responses (cell morphology, cellular junctional plasticity) within a well-plate format and separately study the formation and function of capillary-like lymphatic vascular structures using a unique microfluidic device. We first showed that PhotoCol® could achieve physiologically relevant stiffness values ranging from normal to pathological tissue stiffness levels (~0.5 – 6 kPa shear storage modulus) by altering the photoinitiator solution used for photo-crosslinking (photoinitiators: Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP), Irgacure 2959 (IRG), and Ruthenium/Sodium Persulfate (Ru/SPS)). Results showed that Ru/SPS offered the greatest dynamic stiffness range and highest overall stiffness compared to other photoinitiators, producing stiffness values that allowed us to systematically study human dermal lymphatic endothelial cell (HDLEC) responses related to capillary growth (i.e., morphology) and function (i.e., vascular endothelial (VE)-cadherin cellular junction formation). Our quantitative morphological analysis demonstrated our ability to produce HDLECs with a fibrotic phenotype—larger with more irregular borders and increased VE-cadherin thickness (junction zippering). Subsequently, we investigated the effects of these physiologically relevant stiffnesses on lymphangiogenic sprouting within a microfluidic device. The design of this device (established by Wang et al., 2020), unlike similar microfluidic devices, allows for full exposure of HDLECs to the ECM for studying direct sprouting and migration into the ECM, as well as cellular junctions and vessel permeability. Stiffer ECMs supported increased vascular stability and sprouting of capillary-like structures, while HDLECs within softer ECMs were more migratory with decreased cellular junction formation. This result highlights the role of ECM stiffness in directing the balance between migratory and proliferative phenotypes during vessel formation, which further dictates lymphatic capillarity integrity in normal and disease states. Finally, the underlying mechanisms by which ECM stiffness influences LEC junctional plasticity and subsequent barrier integrity was investigated by modulating LEC mechanosensing via yes-associated protein (YAP) inhibition and vascular endothelial growth factor (VEGF)-A and VEGF-C binding to VEGFR2 and VEGFR2. Overall, this work increases the field’s understanding of the unique contributions of tissue stiffness to lymphatic capillary growth and function under conditions that better reflect the fibrotic environment observed in disease states. By developing an in vitro system that is capable of producing fibrotic LEC and vascular phenotypes, we can move toward future work that investigates ways to effectively target lymphatic vasculature for therapeutic intervention.
| Dissertation Advisor: | Committee Chair: | ||
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Catherine Whittington, PhD Assistant Professor BME Department WPI |
Jeannine Coburn, PhD Associate Professor Biomedical Engineering WPI |
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| Defense Committee: | |||
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Solomon Mensah, PhD Assistant Professor Biomedical Engineering WPI |
Qi Wen, PhD Associate Professor Physics WPI |
Brendon Baker, PhD Associate Professor Biomedical Engineering University of Michigan |
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For a zoom link, please email kharrison@wpi.edu