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Cellular and Molecular Bioengineering

Cellular and Molecular Bioengineering Undergraduate Fire Talks

Evaluating the impact of collagen stiffness and inflammatory cytokine exposure on VE-Cadherin and ZO-1 expression in lymphatic endothelial cells

Saturday, October 11, 2025

9:00 AM - 9:05 AM PDT

Location: Room 33B

Presenting Author(s)

AF

Annabelle Feller

Undergraduate Researcher
Worcester Polytechnic Insitute
Chico, California, United States

Co-Author(s)

Stephen Larson, NA

PhD Candidate
Worcester Polytechnic Institute, United States

Primary Investigator(s)

Catherine F. Whittington

Associate Professor
Worcester Polytechnic Institute, United States

Introduction: : In lymphedema, a chronic condition characterized by disrupted lymphatic drainage and fluid retention, microenvironmental changes around lymphatic capillaries perpetuate lymphatic dysfunction (e.g., altered cell junctions) and chronic inflammation with subsequent fibrotic tissue deposition and tissue stiffening. Three-dimensional in vitro modeling enables researchers to systematically introduce biophysical and biochemical factors to replicate these phenomena [1], and our previous work used PhotoCol methacrylated type I collagen with Ruthenium (Ru)/Sodium Persulfate (SPS) and photo-crosslinking to reach tissue stiffness values comparable to chronic fibrotic conditions (~6 kPa) [2]. Lymphatic endothelial cells (LEC) cultured on soft (~1 kPa) and stiff (~6 kPa) PhotoCol® matrices showed significant differences in cell shape and transitions from ‘oak leaf’-shaped cells with discontinuous button-like cell junctions (VE-Cadherin) on soft PhotoCol® to thicker, continuous zipper-like cell junctions on stiff PhotoCol®. These studies were performed without pro-inflammatory cytokine signaling (e.g., TNF-α, IL-6) that occurs in chronic lymphedema and promotes junction discontinuity [3]. Thus, the current study exposes human dermal lymphatic endothelial cells (HDLECs) grown on 3D soft and stiff PhotoCol® matrices to tumor necrosis factor (TNF)-α, an inflammatory cytokine. Our primary objective was to determine whether expression and localization of adherens (VE-Cadherin) and tight junction proteins (ZO-1) were affected by stiffness and TNF-α exposure. We hypothesized that TNF-α exposure would promote junction discontinuity in HDLEC on soft and stiff PhotoCol® matrices despite stiffness-induced junction zippering. We also addressed an additional research gap by expanding LEC junction analyses for more comprehensive quantification of changes in junction expression and LEC interconnectivity.

Materials and

Methods: : PhotoCol® methacrylated type I collagen (8 mg/mL) with Ruthenium (Ru)/Sodium Persulfate (SPS) photoinitiator (2% v/v) (Advanced BioMatrix) were combined then neutralized (final concentration of, ~7.1 mg/mL). Neutralized PhotoCol® was pipetted into ibidi® µ-slides (15 Well 3D; 12 mL/well) and polymerized at 37 °C (60-90 minutes). Polymerized gels were uncrosslinked (“soft”, ~1kPa) or photo-crosslinked for 90 seconds with 405 nm light (“stiff”, ~6 kPa). Primary human dermal lymphatic endothelial cells (HDLEC) (PromoCell) were seeded on top of PhotoCol® matrices (50,000 cells/cm2) and cultured at 37 °C for 3 days to achieve a monolayer. HDLECs were cultured in MV2 media (PromoCell) supplemented with 5% fetal calf serum without Vitamin C or hydrocortisone. On Day 3, samples were exposed to TNF- α (20 ng/mL) for 24 hours.
To visualize VE-Cadherin and ZO-1, samples were fixed, permeabilized, stained with anti-VE-Cadherin and anti-ZO-1 primary antibodies (AlexaFluor 647 and AlexaFluor 488 secondary antibodies), and counterstained with DAPI. Samples were imaged with a Keyence BZ-X800 microscope (20X objective) to generate max projection images from z-stacks. CellProfiler™ was used to isolate VE-Cadherin junction signal to quantify the number of VE-Cadherin objects, branchpoints, and endpoints (Figure 1). Using a previously developed method to evaluate VE-Cadherin junction thickness [2], distance maps were generated using the Vessel Analysis plugin in FIJI (ImageJ) and applied to threshold images from CellProfiler™. A separate CellProfiler™ pipeline was developed to measure fractional intensity of ZO-1 signal across the cell body (4 bins) extending from the center of identified cell body objects (Figure 1).

Results, Conclusions, and Discussions:: Qualitative analysis revealed that TNF-α treatment produced discontinuous VE-Cadherin expression in soft and stiff matrix conditions and increased nuclear localization of VE-Cadherin (Figure 2A), yet VE-Cadherin junction thickness did not differ between untreated soft and stiff matrix conditions (Figure 2B). Our new, expanded quantitative analysis revealed increased junction discontinuity on LEC boundaries in TNF-α groups by measuring the number of VE-Cadherin objects per nuclei. An overall decrease in junctional continuity and LEC interconnectivity in TNF-α groups was captured by the combination of increased number of endpoints in VE-Cadherin outlines and decreased number of branchpoints (Figure 2B). These findings are consistent with prior 2D studies in which exposure to inflammatory cytokines (e.g., TNF-α) resulted in VE-Cadherin cleavage and increased endothelial barrier permeability [3].
Both adherens junction and tight junction proteins mediate lymphatic vessel permeability, but compared to VE-Cadherin, fewer studies evaluate ZO-1 (tight junction) under fibrotic conditions. Qualitative analysis of ZO-1 expression revealed more distinct, continuous outlines of ZO-1 at cell borders in untreated soft and stiff conditions. However, HDLECs exposed to TNF-α showed less consistent tight junction formation with increased cytoplasmic expression (Figure 3A), prompting us to quantify the fraction of signal at the cell border (Bin 4) (Figure 1C, 3B). Differing patterns of VE-Cadherin expression and ZO-1 localization across groups suggest unique responses between adherens and tight junction proteins to mechanical and biochemical changes in the microenvironment.
Overall, this study identified and leveraged multiple new quantitative measures relevant to characterizing LEC responses to fibrosis, particularly towards increased junctional discontinuity and lower cell interconnectivity observed within TNF-α cytokine exposure. The results aligned with our initial hypothesis. Inflammatory conditions cause more disruptions in LEC interconnectivity, and our prior quantification technique for measuring VE-Cadherin junctional thickness was less effective due to an inability to obtain noise free masks of VE-Cadherin cell outlines for consistent diameter measurements. Therefore, the development of improved quantitative assessments for both junction proteins that account for signal continuity, LEC interconnectivity, and changes in localization will further clarify microenvironmental contributions to LEC barrier function and enable improved in vitro modeling of lymphatic dysfunction and lymphedema progression.

Acknowledgements and/or References (Optional): : Acknowledgements: Thank you to Athenia Jones and Dr. Brian Ruliffson. This work was supported by NSF REU grant EEC2150076.

References: (1) Kolarzyk, A.M. et al., Cold Spring Harb Perspect. Med., 2022, 12(9):a041169. doi:10.1101/cshperspect.a041169. (2) Ruliffson, B.N.K. et al., Lymphatics, 2024, 2(3):177-194. doi: 10.3390/lymphatics2030015. (3) Kraus, S. & Lee, E., Lab Chip, 2023, 23(24):5180:5194. doi: 10.1039/d3lc00486d.