This ERI project investigated how lymphatic capillaries, the smallest lymphatic vessels, respond to changes in their environment, particularly tissue stiffness from fibrosis (excess tissue deposition). Lymphatic vessels are essential for maintaining fluid balance and transporting immune cells, and impaired growth or function occurs in conditions where stiffening tissue affects capillary responses. Lymphatics have been largely understudied regarding tissue stiffness, with prior research focusing on developmental stiffness levels significantly lower than in fibrosis. The ERI project addressed these gaps by using experimental models to explore how tissue stiffness affects cell behaviors related to lymphatic growth and function, which is crucial for advancing lymphatic biology and therapeutic development.

Lymphatic Endothelial Cells Respond to Tissue Stiffness

We developed an in vitro lab-based model using engineered collagen (PhotoCol®) that can form gels at stiffnesses that mimic “normal” and “fibrotic” states upon light exposure. As lymphatic endothelial cells (LEC) grew on these gels, we conducted extensive measurements of cell shape changes (growth indicator) and the thickness of cell-cell junctions (VE-Cadherin protein expression; function indicator). This robust analysis addressed a gap in lymphatic research where quantitative analysis and data on cell shape and junctions was limited.

Results showed that LECs grew larger with thicker cell-cell junctions on stiffer collagen, resembling lymphatic capillaries in fibrotic tissue. Thicker junctions correlated with cell-cell “zippering,” which typically lowers lymphatic capillary function (i.e., movement of cells/molecules across capillary wall), while thinner junctions were “button-like” and typical of LECs in softer, normal tissue. LECs on stiffer collagen also had smoother boundaries with increased elongation, contrasting with the characteristic irregular oak leaf shape seen in LECs in normal conditions. These differences in junction formation were noteworthy, as most studies focus on cell growth more than function.

Since LECs responded to mechanical changes, we examined molecules involved in sensing (YAP) and growth signaling (receptors: VEGFR2, VEGFR3) in LECs. Inhibiting YAP signaling led to fewer cell-cell junctions and increased boundary irregularity, suggesting that YAP may be partially involved in junction formation. Although VEGFR2 and VEGFR3 are typically associated with LEC growth and sprouting, we observed differences in junction formation with receptor inhibition. VEGFR2 inhibition notably decreased VE-Cadherin expression more than VEGFR3 inhibition, indicating a greater role for VEGFR2 in function.

Stiffness Alters Lymphatic Capillary Sprouting

The ERI project utilized a microfluidic device to study lymphatic capillary sprouting in environments with fibrotic stiffness and similar density. The device featured two parallel channels (300 microns wide) within a PhotoCol® gel—one lined with LECs and the other containing molecules to promote LEC sprouting. Over 14 days, LECs sprouted into stiffer collagen, forming short, dense vessel-like structures like those in chronic fibrosis. In softer collagen, most LECs migrated as single cells and some formed discontinuous sprouts near the channel. LEC migration seemed to outpace LEC growth and stabilization, which limited junction formation. Cells lining the channel displayed distinct characteristics similar to what is observed in the body: larger, irregularly shaped cells with jagged junctions (buttoned) in soft collagen versus elongated, uniformly junctioned LECs (zippered) in stiff collagen. Overall, we established a modeling approach to observe differences in sprouting that resembled capillary sprouting fibrotic tissue.

Key Contributions

A major achievement of this work was establishing two experimental lymphatic models that leveraged PhotoCol® as a tunable material to study LEC behavior at fibrotic tissue stiffness levels. We showed that LECs approximated fibrotic responses through changes in cell shape, junction formation, and sprouting patterns. Our ability to discern differences in LECs was enhanced by expanding our analysis beyond standard descriptive assessments to establish robust quantitative measures of cell responses. These findings offer insights into controlling lymphatic capillary growth and function when the surrounding mechanical environment changes. This improved understanding of lymphatic behavior, combined with our robust modeling approaches, lays a foundation for future lymphatic research to address fundamental questions and therapeutic development.

Broader Impacts

The ERI award supported significant education and outreach efforts. The Principal Investigator led several outreach classes and short sessions on biomaterials for high school students, including those in Worcester Polytechnic Institute’s Frontiers STEM Exploration program. Throughout the award term, ~55 students participated in hands-on learning about natural and synthetic materials used in tissue engineering, regenerative medicine, and disease modeling. Through real-world examples, students learned about materials used in the ERI project and other established and emerging biomaterials, fostering early interest in biomedical engineering and biomaterials.

The Principal Investigator also advised undergraduate and graduate students on the ERI project. In addition to lab trainees, senior capstone design teams developed microfluidic chips to support lymphatic capillary growth for applications of their choosing. Undergraduate teams refined chip designs and evaluated material properties while gaining exposure to advanced engineering and biology techniques utilized in the ERI project. Integrating the ERI project into project advising cultivated mentorship between graduate and undergraduate researchers, providing a well-rounded experience in biomedical engineering education.

Last Modified: 05/31/2025
Modified by: Catherine Faye Whittington