Modeling lymphangiogenesis: Pairing in vitro and in vivo metrics

Abstract

Lymphangiogenesis is the mechanism by which the lymphatic system develops and expands new vessels facilitating fluid drainage and immune cell trafficking. Models to study lymphangiogenesis are necessary for a better understanding of the underlying mechanisms and to identify or test new therapeutic agents that target lymphangiogenesis. Across the lymphatic literature, multiple models have been developed to study lymphangiogenesis in vitro and in vivo. In vitro, lymphangiogenesis can be modeled with varying complexity, from monolayers to hydrogels to explants, with common metrics for characterizing proliferation, migration, and sprouting of lymphatic endothelial cells (LECs) and vessels. In comparison, in vivo models of lymphangiogenesis often use genetically modified zebrafish and mice, with in situ mouse models in the ear, cornea, hind leg, and tail. In vivo metrics, such as activation of LECs, number of new lymphatic vessels, and sprouting, mirror those most used in vitro, with the addition of lymphatic vessel hyperplasia and drainage. The impacts of lymphangiogenesis vary by context of tissue and pathology. Therapeutic targeting of lymphangiogenesis can have paradoxical effects depending on the pathology including lymphedema, cancer, organ transplant, and inflammation. In this review, we describe and compare lymphangiogenic outcomes and metrics between in vitro and in vivo studies, specifically reviewing only those publications in which both testing formats are used. We find that in vitro studies correlate well with in vivo in wound healing and development, but not in the reproductive tract or the complex tumor microenvironment. Considerations for improving in vitro models are to increase complexity with perfusable microfluidic devices, co-cultures with tissue-specific support cells, the inclusion of fluid flow, and pairing in vitro models of differing complexities. We believe that these changes would strengthen the correlation between in vitro and in vivo outcomes, giving more insight into lymphangiogenesis in healthy and pathological states.

Keywords: in vitro models; lymphatics; regenerative medicine.

Figure 1.. An overview of components of lymphangiogenesis and relevant in vitro models

Lymphangiogenesis is characterized by proliferation, migration, and sprouting. In vitro models of varying complexity can be employed to investigate these processes. Quantitative metrics often extracted from such assays include percent proliferation, migration and invasion, the distance of migration, and length and number of new vessels or sprouts.

Figure 2.. Hallmarks of lymphangiogenesis in vivo include new vessels, hyperplasia, sprouting, or activation.

In vivo models of lymphangiogenesis typically use genetically modified zebrafish and mice. Some examples of in situ mouse models are the cornea, ear, hind leg, and tail. Histology and lymphatic drainage are used to quantify lymphangiogenesis.

Figure 3.

Lymphangiogenesis in development and wound healing. Dorsal skin from wild-type (WT) and macrophage knockout (PU.1^−/−) embryos is stained for Neuropilin-2 (NRP2 - cyan) for lymphatics and phosphohistone 3 (PH3 - red) for proliferating cells (A). Branch points are denoted by white dots and arrows point to proliferating LECs. Scale bar is 150 μm in 1,2 and 100 μm in 3,4. Branching (B) and proliferation (C) are quantified, showing enhanced proliferation in macrophage knockout mice. In vitro, co-culture of primary embryonic dermal LECs with macrophages causes upregulated proliferation (D). Reproduced with permission from Gordon et al. Wild type (WT) and lymphatic-associated epsin-deficient (LEC-iDKO) mice are fed normal chow or given streptozotocin and high fat diet (STZ/HFD) and lymphangiogenesis in the cornea is demonstrated with LYVE-1 staining (E). Proliferation of LECs is visualized via EdU stain (F). In vitro, primary LECs from each mouse type are cultured on Matrigel for 16 hours, with or without 100 ng/mL VEGF-C for 12 hours (G). Scale bars are 100 μm (E), 20 μm (F), and 50 μm (G). Reproduced with publisher permission from Wu et al.

Figure 4.. Tumor-related lymphangiogenesis.

In vivo, inhibiting fibroblast growth factor receptor signaling decreases lymphatic vessel density. 66c14 transfected mammary tumors from the control (Ctrl), dominant negative FGFR (FGFR-2DN), and parental (Par) groups are stained with VEGFR-3 and nucleus cell marker DAPI (A), vessel density is quantified for each group and shows a significant difference between FGFR-2DN and the Ctrl and Par group (B). In vitro, tube formation assays show decreased tubulogenesis in the FGFR-2DN-expressing clones (C4 and C22) tumor cells cultured with human dermal lymphatic microvascular endothelial cells (C). Scale bars are 200 μm. *p<0.05 versus respective control groups. Reproduced with open access from Larrieu-Lahargue et al. In vivo, TNF-α related lymphangiogenesis is abolished by VEGFR3 blocking and Tnfr1 knockout. Mouse corneas are stained for LYVE-1 (lymphatics) and CD31 (blood vessels) after treatment with VEGF-C, TNF-α, TNF-α and VEGFR3 blocking antibody, or TNF-α and Tnfr-1 knockout (D). In vitro, TNF-α causes alignment of LECs. This is prevented by blocking TNFR1, but not VEGFR-3 (E). Scale bars are 200 μm (D) and 100 μm (E). Reproduced with publisher permission from Ji et al.

Figure 5:. The complexity of in vitro models should increase to better compare in vitro and in vivo outcomes.

Neighboring cells and interstitial fluid in the microenvironment are critical factors to recapitulate. Some considerations are: 1) using 3-dimensional models that allow for sprouting or using a hydrogel that replicates the ECM and includes support cells (i.e. fibroblasts) and immune cells (i.e. macrophages). 2) including different types of fluid flow to better mimic in vivo conditions since lymphatics play key roles in fluid transport and experience both shear stress and interstitial fluid flow in the body. 3) moving towards microfluidic devices that are perfusable to incorporate the benefits of a controlled 3D microenvironment, fluid flow, and allow for functional assessment (i.e. immune cell trafficking, barrier function) of new lymphatic vessels.