The role of soluble growth factors and inhibitors in the vascularisation of lymphoid organs

Lymph nodes contain complex vascular networks composed of lymphatic and blood vessels. The blood vasculature contains specialised high endothelial venules functioning to permit the efficient entry of blood-borne lymphocytes into the node, while the lymphatics contain antigen-presenting cells draining from tissues. In contrast to the well understood cellular interactions and signalling mechanisms driving development of the stromal networks upon which immune cell interactions occur, the processes by which the complex vascular networks develop are poorly characterised. This study aimed to determine the mechanisms by which vascularisation of lymph node anlagen occurs during development. The structure of developing lymph nodes was studied using confocal microscopy to observe the organisation of basement membranes and the different cell types involved in the process. Expression of angiogenesis-related genes was studied using quantitative real-time PCR and microarrays. No evidence for vascularisation of the anlagen was found, though the markers used may not have stained nascent vessels. The lymph sac surrounding the anlagen was shown to exist as a two-layered structure composed of anastomosing blood and lymphatic endothelium. In vitro models of vasculogenesis and angiogenesis were developed utilising human umbilical vein endothelial cells in three dimensional collagen gels, with and without smooth muscle coverage. A spheroid-based model of angiogenesis was used to study the net angiogenic environment in developing E14.5–E17.5 anlagen, which determined that a net anti-angiogenic environment existed at all timepoints in the lymph nodes and thymus, but not skin. Additionally, tensional forces were observed to affect angiogenic sprouting in addition to soluble growth factors. As a consequence of the double-layered lymph sac observed in vitro, and the influence of anti-angiogenic factors and tensional forces observed ex vivo, a model of lymph node development involving anlagen patterning and vascularisation as a result of condensation-induced tensional forces was proposed, complementary to soluble growth factor-driven angiogenesis.