Recent studies on lymphatic vessel formation have mainly focused their interest on organism development. On the contrary, much less is known about the process of lymphangiogenesis occurring in pathological conditions. This study sought to define the ultrastructural features of neo-formed lymphatic vessels and exploited the attributes of two established models of inflammation accompanied by a robust lymphangiogenesis [35
], and the advantage of the recently set up model of lymphatic ring assay which recapitulates all steps of sprouting lymphangiogenesis [38
]. Here, we propose a model of lymphatic vessel formation through tunneling (Figure ). This concept is supported by similar TEM observations generated in three distinct models demonstrating that the formation of lymphatic neo-vessels relies on the alignment of LEC which drive a tunnel through extracellular matrix. During lymphangiogenesis, cords of cells create an extracellular space by the degradation of collagen fibrils occurring extracellularly and intracellularly. Sprouting LEC are characterized by (1) the extension of long thin vacuolized processes which probe the extracellular environment (Figure ), connect with adjacent cells resulting in the formation of cord-like structures and pre-lymphatic vessels consisting in thin tubular structures lined with elongated LEC (Figure ); (2) an intense intracellular vacuolization associated with vesicle coalescence leading to an intracellular luminal space (Figure ); (3) a matrix remodeling generating space between cells promoting cell migration and contributing to lumen formation (Figure ). Furthermore, the present study underlines the strength of the in vitro
lymphatic ring assay which recapitulates the processes observed in vivo
in pathological conditions.
Figure 8 Tunneling model of lymphatic vessel formation. The model is based on ultrastructural observations performed in in vitro and in vivo models of lymphangiogenesis. (A): LEC alignment. Elongated LEC migrate and extend long cytoplasmic protrusions. (B): Vacuolization (more ...)
Emerging descriptions of cellular and molecular events of tubulogenesis occuring during blood vessel formation have converged on three mechanisms underlying angiogenesis: budding (or sprouting), cord hollowing and cell hollowing [19
]. Progress in understanding such angiogenic tube morphogenesis has benefited from 3D culture systems. The present study represents the first ultrastructural description of capillary formation during pathological lymphangiogenesis. In line with the previous descriptions of the angiogenic process, we observed intracellular and extracellular hollowing events. A common feature of the three lymphangiogenic processes studied here is the migration of cells creating spaces that can be occupied by a cord of very thin and elongated cells delimitating a luminal space. In the present study, the involvement of cell proliferation has also been evidenced during cord formation. Cell hollowing or intracellular vacuolization is a mechanism by which individual cells generate vesicles that can enable the cells to interconnect with neighboring cells to form multicellular lumens and tubes [46
]. Cell vacuolization is a common feature of migrating cells in the three models presented here. Vesicles of various sizes were seen to progressively enlarge and fuse to each other to, in turn, form a large intracellular luminal vesicle. By analogy with the angiogenic process, this space likely fuses with vesicle of adjacent cells to form the lumen of a pre-lymphatic vessel. This concept is supported by the process of cell fusion leading to increased lumen size clearly seen in the lymphangioma both at ultrastructural and histological levels (Figures and ). The intracellular vacuolization mechanism was initially associated with the morphogenesis of single endothelial cells which had no contact with adjacent cells occurring during the process of vasculogenesis [44
]. The intracellular vacuolization has been extensively studied in tubulogenesis assay on 3D matrix leading to the identification of key molecular regulators such as matrix metalloproteinases and small GTPase [46
]. In this context, the zebrafish system was suitable to demonstrate the importance of such process in an in vivo
context during developmental conditions [48
]. The present ultrastructural investigation provides the first evidence of intracellular vacuolization in vivo
during lymphangiogenesis. Further investigations are required to give new molecular insights on how this process contributes to lumen formation in lymphatic capillaries. Despite further attempts, we have been unable to set up a real-time visualization of living cells with confocal or two photons microscopes in the lymphatic ring assay.
An exciting advance in the field of angiogenesis came from the finding that several types of specialized endothelial cells (tip cells and stalk cells) are involved in the building of functional blood capillaries. It has been described that lymphatic tip cells expressed more vascular endothelial growth factor receptor-3 (VEGFR3) and neuropilin-2 [52
] but the transposition of the new concept of tip/stalk cells from angiogenic sprouts to lymphangiogenic sprouts in terms of cell proliferation is still premature. In order to shed some light on this issue, we have analyzed the proliferation rate of migrating cells in sprouting capillaries, both in vivo
in the corneal assay, and in vitro
in the lymphatic ring assay. Proliferation assessed by BrdU incorporation was observed both in extending capillaries and at their extremities. In the aortic ring that mimicks the angiogenic process, a quantitative analysis of proliferating cells revealed that none of the tip cells had incorporated BrdU, while 12 ± 5% of the stalk cells were BrdU positive (data not shown). These data suggest that the concept of tip cells defined as non proliferating cells probing the environment can not be extended to the process of lymphangiogenesis and emphasizes differences between the cellular mechanisms underlying lymphangiogenesis and angiogenesis.
Of great interest is our finding that LEC create in vivo
, physical spaces within the surrounding collagen rich environment. This is associated with an extensive extracellular matrix remodeling both evidenced extracellular and intracellularly. Long processes extended by LEC were seen to roll up to enclose matrix fragments and create extracellular spaces. The contribution of MMPs in this remodeling process is supported by the inhibition of LEC sprouting achieved by using a synthetic MMP inhibitor. Such observation is in line with our recent identification of the metalloproteinase-2 (MMP2) which displays collagenolytic activity [53
] as a key regulator of lymphangiogenesis [38
]. Indeed, the embedding of lymphatic duct fragments issued from MMP2-deficient mice led to impaired LEC sprouting and lymphangiogenic response [38
]. The involvement of MMP-driven proteolysis in the lymphangiogenic process is further supported by our previous work using broad spectrum MMP inhibitors in the corneal assay [34
]. It is worth noting that intracellular vacuolization and extracellular remodeling are not two exclusive mechanisms (Figure ). They have been both evidenced in the three distinct in vitro
and in vivo
models used here and thus likely operate concomitantly during lymphangiogenesis. Altogether, our data emphasize the interest of the lymphatic ring assay to unravel the cellular and molecular mechanisms of lymphangiogenesis. It appropriately recapitulates in vitro
the different steps of lymphangiogenesis observed in animal models such as corneal lymphangiogenesis and lymphangioma. The novel emerging panel of in vitro
and in vivo
models of lymphangiogenesis [13
] are suitable to investigate the biology of lymphangiogenesis. This is mandatory for the understanding of several pathological processes such as lymphedema, graft rejection and metastatic dissemination through the lymphatic way.