TABLE 4.

Anti-lymphangiogenic agents explored in pre-clinical studies.

Therapeutic agent	References	Context and mechanism of action
Atorvastatin	Ogata et al., 2016	In the context of an early lymphedema murine model, interactions between TH1/TH17 CD4 + T lymphocytes and macrophages result in increased macrophage VEGF-C expression. Daily oral atorvastatin for 1 month reduces the proportion of IFN-γ– and IL-17–secreting CD4 + T lymphocytes, thereby decreasing VEGF-C expression in lesional macrophages. This therapeutic intervention suppresses pathological lymphangiogenesis that exacerbates lymphedema pathology. The authors describe statins as inhibitors of isoprenoids synthesis, thereby resulting in decreased T cell proliferation and differentiation; however, they concede that the exact therapeutic mechanism of atorvastatin in lymphedema is likely still unknown.
Doxycycline	Han et al., 2014	The authors explored the effects of doxycycline on a corneal inflammation-induced lymphangiogenesis murine model. Topical doxycycline application over a 10-day period post corneal injury resulted in dramatically reduced lymphangiogenesis as compared to control mice. Doxycycline was determined to exert its anti-lymphangiogenic effects via overall inhibition of VEGF-C to VEGFR-3 signaling. Additionally, doxycycline application resulted in reduced VEGF-C–induced human dermal LEC proliferation as well as reduction of macrophage-produced lymphangiogenic factors. The authors deduced that the effects of doxycycline were mediated through the PI3k/Akt pathway and by inhibition of matrix metalloproteinases.
TH1 Cytokines	Shao and Liu, 2006; Kataru et al., 2011	Shao and Liu, 2006 utilized a porcine thoracic duct assay to demonstrate the anti-lymphangiogenic effects of interferon-α and interferon-γ in vitro. These cytokines, produced by TH1 CD4 + T lymphocytes, were deemed to induce LEC apoptosis. Kataru et al., 2011 demonstrated the anti-lymphangiogenic effects of TH1 signaling on lymph nodes through the transfer of T cells to athymic nude mice. Although interferon-γ signaling was identified as a likely mediator of the anti-lymphangiogenic effects of TH1 cells, the authors noted that other T-cell–derived factors may also exert anti-lymphangiogenic effects. Kataru et al., 2011 identified JAK1-STAT1 signaling as a key pathway in interferon-γ lymphangiogenesis inhibition.
TH2 Cytokines	Savetsky et al., 2015; Shin et al., 2015	TH2 CD4 + T lymphocyte-derived cytokines, namely IL-4 and IL-13, were seen to exert anti-lymphangiogenic effects. IL-4 and IL-13 resulted in the downregulation of essential LEC transcription factors. In an in vitro co-culture system of TH2 cells and LECs, TH2 cytokines were seen to inhibit lymphatic tube formation. Shin et al., 2015 demonstrated a therapeutic application of this finding in vivo by inhibiting IL-4 and IL-13 in a murine allergic asthma model; neutralizing antibodies against IL-4 and IL-13 resulted in increased lymphatic vessel density, enhanced functioning of lung lymphatic vessels, and subsequent improvement in antigen clearance.
IL-17A	Chen et al., 2010; Park et al., 2018	In the context of TH17-mediated immune responses, Park et al., 2018 described the anti-lymphangiogenic effects of IL-17A, a cytokine secreted by TH17 CD4 + T lymphocytes. The authors utilized a cholera toxin inflammation model and demonstrated that IL-17A suppresses lymphatic markers in LECs as well as inhibits lymphangiogenesis in the resolution phases of inflammation. The authors then utilized an IL-17A–neutralizing antibody to demonstrate increased lymphangiogenesis and lymphatic function. In a separate study pertaining to non-small cell lung cancer however, Chen et al., 2010 found that IL-17 upregulates VEGF-C expression in cancer cells and subsequently increases tumor lymphangiogenesis. The authors noted that the Lewis Lung carcinoma cells utilized express IL-17 receptor; therefore, it is possible that IL-17 may have context-dependent effects on lymphangiogenesis.
TGF- β	Clavin et al., 2008; Oka et al., 2008	In an acute lymphedema model of the murine tail by Clavin et al., 2008, TGF-β1 expression was modulated via the application of a topical collagen gel. Tail wound repair with collagen gel application resulted in decreased TGF-β1 expression, which in turn resulted in accelerated lymphatic vessel formation and improved healing. The authors found TGF-β1 to have dose-dependent effects on decreasing proliferation and tubule formation of LECs. A separate study by Oka et al., 2008 found that TGF-β reduced expression of LEC markers and lymph vessel development even in the presence of the pro-lymphangiogenic ligand VEGF-C. They also found that TGF-β1 plays important roles in negatively regulating lymphangiogenesis.
VEGFR-3 Blockade	He et al., 2002	As VEGF-C/VEGFR-3 signaling is the main driver of lymphangiogenesis, blockade of this interaction has long been utilized to exert anti-lymphangiogenic effects. He et al., 2002 transfected human lung cancer cell lines with a soluble fusion protein VEGFR-3-immunoglobulin and then implanted these tumor cells subcutaneously into severe combined immunodeficient mice. The soluble VEGFR-3 protein expressed by tumor cells inhibited VEGF-C/VEGFR-3 interaction. Through this experiment, the authors determined that inhibition of the VEGF-C/VEGFR-3 interaction can suppress tumor lymphangiogenesis and thereby prevent metastasis to regional lymph nodes; however, metastasis of tumor cells to the lungs still occurred via factors extraneous to VEGF-C signaling.