Investigating lymphatic vascular remodelling during postnatal mouse mammary gland morphogenesis.

Abstract

The lymphatic vasculature, an essential component of the cardiovascular system, serves several functions critical to embryonic development and adult homeostasis. Lymphatic vessels return interstitial protein-rich fluid to the bloodstream, transport immune cells during immune surveillance and infection and absorb lipids from the digestive tract (Alitalo et al., 2005; Tammela and Alitalo, 2010). The aberrant growth and development of lymphatic vessels (lymphangiogenesis) is a common feature of human disorders including lymphoedema, inflammatory diseases and tumour metastasis (Alitalo et al., 2005; Tammela and Alitalo, 2010). Lymphatic vessels are of key importance to breast cancer patients. Lymphatic vessels are exploited as a key route of metastasis for tumour cells and the ability of a tumour to promote lymphangiogenesis has been linked with metastasis and poor patient prognosis (Gu et al., 2008; Nakamura et al., 2005; Nakamura et al., 2003; Ran et al., 2010; Skobe et al., 2001). Moreover, lymphatic vascular damage incurred during the surgical resection of lymph nodes commonly results in secondary lymphoedema, a debilitating complication for up to 40% of breast cancer patients (Armer et al., 2009). Despite the involvement of lymphatic vessels in breast cancer, the genes and molecular mechanisms that regulate lymphangiogenesis in the breast remain relatively uncharacterised. The mammary epithelium and blood vasculature undergo dynamic remodelling events in response to hormonal signals and functional demands during postnatal mouse mammary gland morphogenesis (Djonov et al., 2001; Matsumoto et al., 1992; Richert et al., 2000; Watson and Khaled, 2008). The aims of this project were: 1. To investigate the spatial organisation of lymphatic vessels in the mouse mammary gland. 2. To investigate whether lymphatic vessels, like blood vessels and the mammary epithelial tree, are temporally remodelled during mouse mammary gland morphogenesis. 3. To define signals that regulate lymphangiogenesis during postnatal mouse mammary gland morphogenesis. This study provides the first evidence demonstrating that the lymphatic vasculature is dynamically remodelled along with the mammary epithelial tree and blood vasculature during postnatal mouse mammary gland morphogenesis. In addition, this study reveals an intimate association of lymphatic vessels with epithelial ducts, a finding that has important implications for tumour metastasis, as well as the spatial organisation of lymphatic vessels in other branched epithelial tissues, including the lung, kidney, pancreas and prostate. Furthermore, we established that vascular endothelial growth factor (Vegf) C (Vegfc) and Vegfd mRNA levels are significantly increased early during pregnancy and that proteolytically-processed, active VEGF-D is expressed selectively in pregnant, but not virgin mouse mammary glands, corresponding with the stage of peak lymphatic vessel density. In accordance with these data, we demonstrated that a tyrosine kinase inhibitor specific for VEGF receptor 3 (Kirkin et al., 2001; Kirkin et al., 2004), the principal receptor for mouse VEGF-C and VEGF-D, can block the proliferation of primary dermal lymphatic endothelial cells that is stimulated by mammary epithelial and stromal cell conditioned media ex vivo. These data suggest that VEGF-C and VEGF-D, two of the best characterised lymphangiogenic stimuli to date, are likely to play key roles in the stimulation of lymphangiogenesis in the pregnant mouse mammary gland. Elucidation of the molecular mechanisms controlling lymphangiogenesis in the mammary gland has the potential to reveal important targets for the future generation of pro- and anti-lymphangiogenic therapeutics, with the ultimate goal to repair surgically damaged lymphatic vessels and prevent breast cancer metastasis, respectively.