Fibroblast cells regulate lymphatic endothelial cell junctional integrity, modulating lymphatic transport

Introduction: The lymphatic vasculature plays a critical role in draining interstitial fluid from tissues, repairing tissues, and trafficking immune cells. Lymphatic endothelial cells (LECs) line lymphatic vessels and are responsible for transporting fluid across vessel walls. Cell-cell junctions control barrier function (i.e. vascular permeability) including transport of peripheral tissue fluids that include proteins, minerals, fats, cell debris and germs. Lymphatic capillaries are lined with button-like junctions, while lymphatic collective vessels are lined with zipper-like junctions making them less permeable [1]. Tight junction proteins, such as zonula occludens (ZO-1) and claudin 5 are attached to actin fibers and work with adherens proteins, like vascular endothelial (VE)-cadherin to maintain vascular integrity and barrier function [2]. Lymphatic dysfunction may lead to dysregulated transport across lymphatic vessels and accumulation of lymph, which occurs in several diseases, including lymphedema, inflammatory bowel disease, Alzheimer’s, and multiple sclerosis [3,4]. Fibroblasts are important cells to constitute the lymphatic interstitial environment by secreting extracellular matrix (ECM) proteins and affecting lymphatic functions and lymphangiogenesis [5-7]. However, the direct effect of fibroblast cells on lymphatic barrier function and integrity is not well defined. Here, we developed an in-vitro co-culture model to examine regulation of LEC junctional morphology and permeability by fibroblasts.

Materials and

Methods: Normal human lung fibroblast (NHLF) and normal human dermal fibroblast (NHDF) cells were cultured with monolayers of primary human dermal lymphatic cells (HDLEC) for noncontact in-vitro co-culture assay. HDLECs were either treated with preconditioned media collected from fibroblasts or were co-cultured with fibroblast cells in transwell inserts. Integrity of the HDLEC monolayer was verified via trans-endothelial electrical resistance (TEER). HDLEC cell-cell junction protein expression and morphology including ZO-1, VE-cadherin, claudin-5, and cytoskeletal actin fibers were determined by immunofluorescence staining and confocal imaging. Images were processed with FIJI and a special junction analyzer program (JAnaP) [8]. The JAnaP algorithm identifies junction phenotypes and categorizes them into either punctate, perpendicular, or continuous junctions. The program further quantifies junction area, solidity, and circularity. Further, we investigated HDLEC permeability after exposure to fibroblast preconditioned media or co-culture by assessing the transport of 4kDa and 150kDa Dextran. Dextrans were placed on the apical side of the transwell and the bottom, basolateral compartment was sampled, and fluorescence intensity was measured to determine the amount of material transported over time.

Results, Conclusions, and Discussions:

Results:
HDLEC monolayer integrity in the presence of NHLF preconditioned media (NHLF soup) was tested and found that despite an increase in expression of junctional proteins there was a significant decrease in HDLEC TEER (Figure 1). Additionally, when we increased soup proportions (from 10% to 50%) transportation of 4kDa dextran (FD4) across HDLEC increased. In contrast, when we exposed HDLECs to NHDF preconditioned media (NHDF soup), the opposite happened with an increase in TEER values. Moreover, exposing HDLECs to NHDF soup resulted into high expression of junction proteins including ZO-1, claudin-5, and VE-cadherin and increased formation of actin stress fibers (Figure 2). The more direct impact of the NHDF cells on HDLEC junctions was also investigated with a transwell co-culture system. We found that co-culture increased expression of ZO-1 and VE-cadherin in HDLECs compared to untreated cells (Figure 3). The transport assay supported our observation that junction morphology suggested a tighter HDLEC barrier in the presence of NHDFs, as we saw a decrease in FD4 and Dextran 150 kDa transport across monolayers in NHDF co-cultured endothelial cells, suggesting that NHDFs promoted tight junctions. Furthermore, junction morphology analysis showed fewer punctate junctions and a more continuous junction phenotype between HDLECs in the presence of NHDF cells (72%) than control HDLEC (33%).

Conclusions:
Research has suggested that the barrier function of LECs is directly affected by their interstitial tissue microenvironment [9,10]. Here, we saw that the origin of fibroblasts highly influenced HDLEC permeability. When treated with dermal fibroblast-conditioned media or co-cultures, HDLECs showed increased junctional ZO-1, claudin 5, and VE-cadherin expression, suggesting improved barrier maturation. We also found that treatment with NHDFs reduced material transport across the HDLEC monolayer, thus improving endothelial integrity.
Discussions:
The current study highlights the effect of fibroblast cells on LEC morphology and cell-cell junctions and suggests their role in regulating lymphatic vessel integrity and barrier function. The study has implications in understanding effective lymph targeting strategy for vaccine delivery and enhanced therapeutic efficacy.

Acknowledgements (Optional):

References:
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