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Lymphangiogenesis is an essential physiological process but also a determining factor in vascular-related pathological conditions. Angiopoietin-2 (Ang2) plays an important role in lymphatic vascular development and function and its upregulation has been reported in several vascular-related diseases, including cancer. Given the established role of the small GTPase RhoA on cytoskeleton-dependent endothelial functions, we investigated the relationship between RhoA and Ang2-induced cellular activities. This study shows that Ang2-driven human dermal lymphatic endothelial cell migration depends on RhoA. We demonstrate that Ang2-induced migration is independent of the Tie receptors, but dependent on β1 integrin-mediated RhoA activation with knockdown, pharmacological approaches, and protein sequencing experiments. Although the key proteins downstream of RhoA, Rho kinase (ROCK) and myosin light chain, were activated, blockade of ROCK did not abrogate the Ang2-driven migratory effect. However, formins, an alternative target of RhoA, were identified as key players, and especially FHOD1. The Ang2-RhoA relationship was explored in vivo, where lymphatic endothelial RhoA deficiency blocked Ang2-induced lymphangiogenesis, highlighting RhoA as an important target for anti-lymphangiogenic treatments., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)

Lymphangiogenesis, the formation of lymphatic vessels from preexisting ones, is an important process in wound-healing physiology. Deregulation of lymphangiogenesis and lymphatic vascular remodeling have been implicated in a range of inflammatory conditions, such as lymphedema, lymphadenopathy, tumor growth, and cancer metastasis. Any attempt in understanding various parameters of the lymphangiogenic process and developing desirable therapeutic targets requires recapitulating these conditions in in vivo models. One pitfall with some experimental models is the absence of immune response, an important regulatory factor for lymphangiogenesis. We overcome this issue by using immune competent mice. In this chapter, by using Angiopoietin-2 (Ang2), a protein that belongs to the Ang/Tie signaling pathway, we describe the ear sponge assay with important adaptations, highlighting a reproducible and quantitative tool for assessment of in vivo lymphangiogenesis.

During the growth of lymphatic vessels (lymphangiogenesis), lymphatic endothelial cells (LECs) at the growing front sprout by forming filopodia. Those tip cells are not exposed to circulating lymph, as they are not lumenized. In contrast, LECs that trail the growing front are exposed to shear stress, become quiescent, and remodel into stable vessels. The mechanisms that coordinate the opposed activities of lymphatic sprouting and maturation remain poorly understood. Here, we show that the canonical tip cell marker Delta-like 4 (DLL4) promotes sprouting lymphangiogenesis by enhancing VEGF-C/VEGF receptor 3 (VEGFR3) signaling. However, in lumenized lymphatic vessels, laminar shear stress (LSS) inhibits the expression of DLL4, as well as additional tip cell markers. Paradoxically, LSS also upregulates VEGF-C/VEGFR3 signaling in LECs, but sphingosine 1-phosphate receptor 1 (S1PR1) activity antagonizes LSS-mediated VEGF-C signaling to promote lymphatic vascular quiescence. Correspondingly, S1pr1 loss in LECs induced lymphatic vascular hypersprouting and hyperbranching, which could be rescued by reducing Vegfr3 gene dosage in vivo. In addition, S1PR1 regulates lymphatic vessel maturation by inhibiting RhoA activity to promote membrane localization of the tight junction molecule claudin-5. Our findings suggest a potentially new paradigm in which LSS induces quiescence and promotes the survival of LECs by downregulating DLL4 and enhancing VEGF-C signaling, respectively. S1PR1 dampens LSS/VEGF-C signaling, thereby preventing sprouting from quiescent lymphatic vessels. These results also highlight the distinct roles that S1PR1 and DLL4 play in LECs when compared with their known roles in the blood vasculature.

Head and neck squamous cell carcinomas (HNSCC) often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. HNSCCs are remarkably lymphangiogenic and represent a clear example of a cancer that utilizes the lymphatic vasculature for malignant dissemination; however, the molecular mechanisms underlying lymphangiogenesis in HNSCC is still poorly understood. Of interest, we found that an axon guidance molecule, Semaphorin 3F (SEMA3F), is among the top 1% underexpressed genes in HNSCC, and that genomic loss of SEMA3F correlates with increased metastasis and decreased survival. SEMA3F acts on its coreceptors, plexins and neuropilins, among which neuropilin-2 (NRP2) is highly expressed in lymphatic endothelial cells (LEC) but not in oral epithelium and most HNSCCs. We show that recombinant SEMA3F promotes LEC collapse and potently inhibits lymphangiogenesis in vivo. By reconstituting all possible plexin and neuropilin combinations, we found that SEMA3F acts through multiple receptors, but predominantly requires NRP2 to signal in LECs. Using orthotopic HNSCC metastasis mouse models, we provide direct evidence that SEMA3F re-expression diminishes lymphangiogenesis and lymph node metastasis. Furthermore, analysis of a large tissue collection revealed that SEMA3F is progressively lost during HNSCC progression, concomitant with increased tumor lymphangiogenesis. SEMA3F is localized to 3p21, an early and frequently deleted locus in HNSCC and many other prevalent human malignancies. Thus, SEMA3F may represent an antilymphangiogenic metastasis suppressor gene widely lost during cancer progression, hence serving as a prognostic biomarker and an attractive target for therapeutic intervention to halt metastasis., (©2015 American Association for Cancer Research.)

Despite our improved understanding of cancer, the 5-year survival rate for head and neck squamous cell carcinomas (HNSCC) patients remains relatively unchanged at 50% for the past three decades. HNSCCs often metastasize to locoregional lymph nodes, and lymph node involvement represents one of the most important prognostic factors of poor clinical outcome. Among the multiple dysregulated molecular mechanism in HNSCCs, emerging basic, preclinical, and clinical findings support the importance of the mTOR signaling route in HNSCC progression. Indeed, we observed here that the activation of mTOR is a widespread event in clinical specimens of HNSCCs invading locoregional lymph nodes. We developed an orthotopic model of HNSCC consisting of the implantation of HNSCC cells into the tongues of immunocompromised mice. These orthotopic tumors spontaneously metastasize to the cervical lymph nodes, where the presence of HNSCC cells can be revealed by histologic and immunohistochemical evaluation. Both primary and metastatic experimental HNSCC lesions exhibited elevated mTOR activity. The ability to monitor and quantitate lymph node invasion in this model system enabled us to explore whether the blockade of mTOR could impact HNSCC metastasis. We found that inhibition of mTOR with rapamycin and the rapalog RAD001 diminished lymphangiogenesis in the primary tumors and prevented the dissemination of HNSCC cancer cells to the cervical lymph nodes, thereby prolonging animal survival. These findings may provide a rationale for the future clinical evaluation of mTOR inhibitors, including rapamycin and its analogues, as part of a molecular-targeted metastasis preventive strategy for the treatment of patients with HNSCC., (©2011 AACR)

Translocation of bacteria and their products across the intestinal barrier is common in patients with liver disease, and there is evidence that experimental liver fibrosis depends on bacterial translocation. The purpose of our study was to investigate liver fibrosis in conventional and germ-free (GF) C57BL/6 mice. Chronic liver injury was induced by administration of thioacetamide (TAA) in the drinking water for 21 wk or by repeated intraperitoneal injections of carbon tetrachloride (CCl4). Increased liver fibrosis was observed in GF mice compared with conventional mice. Hepatocytes showed more toxin-induced oxi-dative stress and cell death. This was accompanied by increased activation of hepatic stellate cells, but hepatic mediators of inflammation were not significantly different. Similarly, a genetic model using Myd88/Trif-deficient mice, which lack downstream innate immunity signaling, had more severe fibrosis than wild-type mice. Isolated Myd88/Trif-deficient hepatocytes were more susceptible to toxin-induced cell death in culture. In conclusion, the commensal microbiota prevents fibrosis upon chronic liver injury in mice. This is the first study describing a beneficial role of the commensal microbiota in maintaining liver homeostasis and preventing liver fibrosis. [ABSTRACT FROM AUTHOR]