Investigating the effect of interstitial fluid flow on nanoparticle transport across lymphatic endothelial cells and their junction integrity

Introduction: Lymphatic vessels are essential for maintaining tissue homeostasis by collecting excess fluid and returning it to systemic circulation. Lymphatic endothelial cells (LECs) line lymphatic vessels, and their cell-cell junction determines vessel permeability. Initial lymphatic vessels are found in peripheral tissues and collect fluid leaking from their blood vessel. Their cell-cell junction is expressed in a discontinuous, button-like manner to allow fluid entry. In contrast, larger collecting vessels guide fluid through filtration into lymph nodes and back to systemic circulation. Larger collecting vessels must have low permeability to prevent leakage into the extracellular space, primarily characterized by their tight zipper-linked cell junctions.
LECs constantly experience mechanical forces, like interstitial fluid flow, influencing lymph uptake and propulsion. Both aging and pathological conditions, such as lymphedema, can cause dysregulation and a decline in lymphatic vasculature and function. This dysfunction impairs interstitial fluid balance and immune response, particularly in collecting vessels, which become leaky.
In this study, we used in vitro models and computational analyses to investigate how transmural flow across LECs and into vessel lumen affects junction morphology and permeability.
Previous work has shown that LEC permeability increases with interstitial fluid flow across vessel lumens in a flow-rate-dependent manner. Here, we quantify and characterize cell-cell junctions to correlate junction morphology and junction protein expression with LEC permeability. Our work provides insights into what may cause dysregulation of lymphatic function and integrity during pathological conditions. Our results can provide targets to modulate lymphatic functions and better treat or prevent lymphatic diseases.

Materials and

Methods: In vitro transport assay: We employed a previously established in vitro permeability assay to stimulate transport into the vessel lumen. LECs are grown on the bottom of transwells (passages 5-8) until confluence and treated with a fluid pressure head mimicking homeostasis (0.1 – 1 µm/s) or inflammatory states (1 µm/s-5 µm/s) of flow (Fig.1A). Monolayer confluence was confirmed via transendothelial electrical resistance and a nuclei/plasma membrane live stain. Nanoparticles, along with fluorescence microscopy and computational analyses, were utilized as tools to study lymphatic permeability. Nanoparticles are placed on LECs to measure uptake and transport with and without flow.
Nanoparticle PEGylation and Characterization: Fluorescently labeled, 100nm carboxyl (COOH)- polystyrene (PS) nanoparticles are used to measure uptake and transport with and without flow. Polyethylene glycol (PEG)-Amine was conjugated to carboxylic acid functionalized polystyrene (PS) nanoparticles via ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) chemistry and analyzed using dynamic light scattering and phase analysis light scattering.
Staining, Imaging, and Quantifying Cell-Cell Junction Morphologies: LECs on transwells were fixed with 2% paraformaldehyde and stained for F-actin, zona occludin-1, and VE-cadherin via immunofluorescence. Junction expression was quantified using confocal microscopy (Olympus Fluoview) followed by analysis via Image J, and junction and nuclear morphologies were analyzed using the Junction Analyzer Program (JAnaP) developed by the Stroka Lab and compared to junctions found in vivo.

Results, Conclusions, and Discussions: Our research has revealed a significant finding: interstitial fluid flow has a profound impact on nanoparticle transport by LECs, increasing it by more than five-fold within a few hours, without compromising monolayer integrity (Fig. 1B). Furthermore, we observed a flow-dependent increase in nanoparticle transport, indicating that LECs are responsive to environmental cues such as interstitial fluid flow, regulating the influx of nanoparticles into the vessel lumen.
To quantify how fluid flow treatment affects LEC junction morphology, cells were treated with flow or static (no flow) conditions overnight (ON) or for two hours (2h) (Fig. 2). We see a difference in flow treatment duration, but not in pressure-driven flow itself. We found that, on average, a 2-hr treatment, regardless of flow treatment (flow and static), resulted in 42% continuous junctions compared to ON treatment (55%) (Fig. 2A). ON treatments show more staggered and disorganized junction expressions (orange arrows) (Fig. 2B and 2C). In contrast, with 2h treatments, there is more zipper-like junction expression (red arrows)(Fig. 2D and 2E). With ON treatment, continuous junction expression decreases by 13% (Fig. 3A) compared to 2hr treatment. Conversely, on average, there is an 18% decrease for 2h treatment in discontinuous and perpendicular junction expression compared to ON treatment (Fig. 3B and 3C). We expected flow treatment to cause more permeable LECs and thus more punctate expression than static. However, we see little to no difference in punctate junction expression between flow treatments (Fig. 3D). We hypothesized flow influences LEC permeability. However, our data suggests that the duration of flow treatment conditions has a larger effect. We aim to optimize JAnaP to button-like and zipper-like junctions in LECs. Overall, flow influences nanoparticle transport across LECS, which we hypothesize to be mediated by paracellular transport mechanisms.

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