TABLE 3.

Animal models for the study of LV regression.

Model	References	Utility and findings
Cornea (mouse)	Zhang et al., 2011; Shi et al., 2020	In tandem, these studies demonstrated the utility of the murine cornea for observing de novo lymphangiogenesis and LV regression. Due to the ordinarily avascular nature of the cornea, Prox-1 GFP transgenic mice can be utilized for live-imaging of LV progression and regression. The latter reference utilized this technology to demonstrate potentially therapeutic properties of aqueous humor in inducing LV regression.
Tail (mouse)	Bramos et al., 2016; Weiler et al., 2019; Zhou et al., 2020; Hassanein et al., 2021	The mouse tail lymphedema model involves 2-mm deep surgical circumferential excision of the portion of the tail 2 cm distal to the tail base, which disconnects the superficial and deep lymphatics of the tail, thereby locally mimicking lymphedema pathology. Visualization of lymphatic flow involves injection of a fluorescently labeled tracker, such as fluorescein isothiocyanate-dextran, into the mouse tail.
Bone (mouse)	Monroy et al., 2020	This work utilized several transgenic mouse models to visualize de novo lymphangiogenesis and LV regression in the context of generalized lymphatic anomaly, a pathology that may be caused by activating mutations of PIK3CA. Prox1-CreERT2;LSL-Pik3caH1047R transgenic mice offer a tamoxifen-inducible system for expression of PIK3CA in LECs. Osx-tTA-TetO-Cre;TetO-Vegfc;mT/mG transgenic mice offer a murine model of Gorham-Stout disease. This model utilizes the bone-specific Osterix promoter to drive a Tet-On system for VEGF-C overexpression in osteoblasts, osteocytes, and chondrocytes. Furthermore, the mT/mG reporter system causes all Cre-positive cells to express GFP. This work highlights the utility of designing transgenic mice to study LV progression and regression in a wide variety of disease contexts.
Lung (mouse)	Yao et al., 2014	CCSP-rtTA; tetO-VEGF-C transgenic mice can be used to study de novo lymphangiogenesis in the context of pulmonary lymphangiectasia. This construct allows for doxycycline-induced expression of VEGF-C in Clara cells and alveolar type II cells. These mice were crossed with Prox1-GFP mice to allow for live imaging of LVs, and the triple transgenic mouse allows for an analysis of the effects of VEGF-C to VEGFR-3 signaling in a pulmonary context. Utilization of this model revealed a critical period when VEGF-C expression and resultant lymphangiogenesis produce a pulmonary lymphangiectasia pathology. This model also enables the study of therapeutic modulation of LV regression to treat and prevent pulmonary lymphangiectasia.
Long-lived human LECs	Frenkel et al., 2021	A microfluidic LV model enables analysis of de novo lymphangiogenesis and tumor–LV interaction. Lentiviral delivery of human telomerase and BMI-1 expression cassettes was utilized to develop an immortalized human LEC line. This cell line was paired with a microfluidic chip consisting of a free-standing extracellular matrix to visualize the formation of LV-like structures. This model was next co-cultured with mouse colon cancer organoids, enabling live visualization of tumor-induced lymphatic vasculature changes. This microfluidic model can be utilized to mimic both native and tumoral contexts of lymphangiogenesis and LV regression. Application of exogenous therapeutics or molecules of interest to this model can be used to study methods for modulating LV regression and lymphangiogenesis.
Lymph Node	Hirakawa et al., 2005; Mumprecht et al., 2012; Truman et al., 2012	Transgenic mice overexpressing VEGF-A were seen to exhibit sentinel lymph node lymphangiogenesis in a cutaneous squamous cell carcinoma model. While this study by Hirakawa et al., 2005 identified VEGF-A as a tumor lymphangiogenesis inducer, it also demonstrated the utility of the lymph node as a model to study lymphatic vessel dynamics. A different study utilized radiolabeled antibodies against LYVE-1 in conjunction with positron emission tomography to visualize murine lymph node lymphangiogenesis in response to induced inflammation of the skin. Upon resolution of inflammation 3 months later, the authors observed LV regression, thereby demonstrating that inflammation-induced lymph node lymphangiogenesis is indeed reversible. With the progression of transgenic fluorescent reporter mice and robust visualization of LVs in lymph nodes, this remains a powerful tool for exploring lymphatic vessel dynamics.