Lymphatic collecting vessel maturation and valve morphogenesis

YMVRE-03448; No. of pages: 7; 4C: Microvascular Research xxx (2014) xxx–xxx

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Lymphatic collecting vessel maturation and valve morphogenesis Daniel Vittet ⁎ Inserm, U1036, Grenoble, F-38000 France, CEA, DSV, iRTSV, Laboratoire Biologie du Cancer et de l'Infection, Grenoble, F-38000 France, Univ Grenoble Alpes, Grenoble, F-38000 France

a r t i c l e

i n f o

Article history: Accepted 3 July 2014 Available online xxxx Keywords: lymphangiogenesis smooth muscle cell lymphatic endothelial cell lymphatic development molecular mechanisms lymphatic capillaries collecting vessels

a b s t r a c t The lymphatic vasculature plays an essential role in the maintenance of tissue interstitial fluid balance and in the immune response. After capture of fluids, proteins and antigens by lymphatic capillaries, lymphatic collecting vessels ensure lymph transport. An important component to avoid lymph backflow and to allow a unidirectional flow is the presence of intraluminal valves. Defects in the function of collecting vessels lead to lymphedema. Several important factors and signaling pathways involved in lymphatic collecting vessel maturation and valve morphogenesis have now been discovered. The present review summarizes the current knowledge about the key steps of lymphatic collecting vessel development and maturation and focuses on the regulatory mechanisms involved in lymphatic valve formation. © 2014 Elsevier Inc. All rights reserved.

Organization and function of the lymphatic vascular system The lymphatic vascular system is a one-way transport system responsible for the maintenance of fluid homeostasis. The lymphatic vasculature is organized into a network of lymphatic vessels that collect extravasated tissue fluid from the interstitial space and drain it back to the venous circulation. This allows the transport of interstitial proteins in excess and waste back to the blood circulation. It is also involved in fat absorption and represents the major route for immune cells and soluble antigens to reach the lymph nodes where immune responses are activated. During development, after the establishment of a primary lymphatic vasculature, lymphatic vessels undergo further remodeling to form a functional lymphatic vessel network consisting in a hierarchical vascular tree composed of lymphatic capillaries, pre-collectors and collecting vessels (Fig. 1). Lymphatic capillaries are blind-ended thin walled vessels. They lack mural cell coverage, are devoid of a continuous basement membrane, and are connected to the extracellular matrix by anchoring filaments (reviewed in (Schulte-Merker et al., 2011)). Fluid uptake in lymphatic capillaries is governed by the increase in interstitial fluid pressure. The entry into the capillaries is facilitated by discontinuous VE-cadherin positive button junctions. The lymph contained in capillaries is then drained into pre-collector lymphatic vessels and further into larger lymphatic collecting vessels. Pre-collectors display similar properties as capillaries with the exception that they also contain bi-leaflet intraluminal valves like those seen in large collecting vessels. Collecting vessels are characterized by the presence of zipper-like ⁎ Inserm U1036, Biology of Cancer and Infection (BCI), iRTSV, CEA Grenoble, 17 rue des martyrs, F-38054 Grenoble Cedex 9, France. Fax: +33 438 78 50 58. E-mail address: [email protected].

junctions between endothelial cells, a continuous basement membrane, smooth muscle cell (SMC) coverage and the presence of valves (reviewed in (Schulte-Merker et al., 2011)). These valves, that are regularly distributed, prevent retrograde flow and ensure a unidirectional lymph flow back to the blood circulation. Collecting vessels are divided into distinct functional units, called lymphangions. A lymphangion corresponds to the part of the vessel located between two valves. They constitute contractile compartments, active lymph propulsion being achieved by the intrinsic contractions of the lymphangions. This is in contrast with what occurs at the level of capillaries where the main identified factors influencing lymph flow, such as the respiratory movements and the surrounding skeletal muscle contractions, are indirect. However, lymphatic contractile mechanisms remain poorly understood. The collecting lymphatic vessels drain into trunks and ducts that connect with the blood vascular system at both right and left subclavian veins and at the left jugular vein which are the major physiological connections between blood and lymph vessels (reviewed in (Margaris and Black, 2012)). The lymphatic vasculature participates in the pathogenesis of several diseases (reviewed in (Tammela and Alitalo, 2010)). Indeed, the impairment of lymphatic drainage resulting from lymphatic vascular insufficiency can lead to lymphedema development. This may occur as a consequence of infection, trauma or surgery that has disrupted lymphatic vessels. It results in a marked swelling of the tissues in the injured region. Lymphedema may also have, in some cases, a congenital origin (reviewed in (Tammela and Alitalo, 2010) and (Schulte-Merker et al., 2011)). Since the collecting lymphatic vessels are connected with the chains of lymph nodes that are essential components of the immune response, the lymphatic vascular system affects the efficiency of the immune function. Finally, lymphatic vessels can also serve as a way for tumor cells to reach lymph nodes and eventually more distant sites and thus promote tumor cell dissemination.

http://dx.doi.org/10.1016/j.mvr.2014.07.001 0026-2862/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Vittet, D., Lymphatic collecting vessel maturation and valve morphogenesis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.07.001

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Fig. 1. Schematic representation of the organization and of the molecular identity of the lymphatic vasculature. The blind-ended lymphatic capillaries capture fluids, proteins and cells from the interstitial space. They are constituted of oak leaf-shaped cells that are connected to the extracellular matrix by anchoring filaments and that display button-like intercellular junctions. Lymphatic endothelial cells (LECs) of the collecting lymphatic vessels that transport the lymph have a basement membrane and exhibit zipper-like intercellular junctions. Collecting vessels are covered by smooth muscle cells (SMCs) that possess intrinsic contractile activity ensuring lymph propulsion. They contain intraluminal valves to prevent backflow. The LECs of capillaries, collecting vessels and valve-forming cells exhibit differences in their molecular identity. Their respective expression profiles are indicated in boxes.

Overview of lymphatic vascular development In the mouse mammalian embryo, it has been demonstrated that the lymphatic vasculature originates from a subset of blood venous endothelial cells of the cardinal veins at E9-9.5 (reviewed in (MartinezCorral and Makinen, 2013), (Koltowska et al., 2013) and (Yang and Oliver, 2014)). The intersomitic veins and the superficial venous plexus have been recently characterized as other locations where can arise lymphatic endothelial cell progenitors (Yang et al., 2012) (Hagerling et al., 2013). Lymphatic competence as defined by cellular expression of LYVE-1 (lymphatic vessel endothelial hyaluronan receptor 1) and VEGFR3 (vascular endothelial growth factor receptor 3) represents the first step towards differentiation into the lymphatic lineage. These lymphatic endothelial cell (LEC) progenitors then migrate out the cardinal veins to form the primary lymphatic sacs. The subsequent expansion of a primary lymphatic vascular plexus is achieved through proliferation and sprouting from these sacs. The specification of lymphatic fate is controlled by key transcription factors: COUP-TFII (Chicken ovalbumin upstream transcription factor II), SOX18 (SRY-related HMG-box 18) and Prox1 (Prospero-related homeobox domain 1), whereas lymphatic endothelial migration and sprouting leading to the primary lymphatic plexus has been shown to be mainly under the control of VEGFC (Vascular endothelial growth factor C) and of its receptor VEGFR3 (Francois et al., 2011; Karkkainen et al., 2004). This primary network further undergoes remodeling and maturation to give a functional

lymphatic network composed of lymphatic capillaries, pre-collectors and collecting lymphatic vessels. This later process starts at E15.5 and continues early after birth. It involves the acquisition of specific characteristics that allow the specialized function of each type of lymphatic vessel (reviewed in (Martinez-Corral and Makinen, 2013)). As a consequence, lymphatic capillaries and collecting vessels display distinct molecular identities (Fig. 1). On the other hand, it is important to note that the characterization of the morphological and functional differences between the lymphatic vasculature of different organs or tissues has been poorly investigated and remains very limited. Lymphatic remodeling and collecting vessel identity specification The specification of collecting vessels is characterized by the deposition of a thin continuous basement membrane, the recruitment of SMCs and the formation of luminal valves. Collecting vessel identity is also characterized by the down-regulated expression of several lymphatic endothelial markers highly expressed in LECs during development and in lymphatic capillaries (Fig. 1). After maturation, LYVE-1 is almost completely lost on endothelial cells of the collecting vessels (Norrmen et al., 2009). Gene targeting experiments in mice have revealed that LYVE-1 deficiency does not significantly affect lymphatic development and function (Gale et al., 2007). Thus, the significance of LYVE-1 down-regulation during collecting vessel maturation remains to be

Please cite this article as: Vittet, D., Lymphatic collecting vessel maturation and valve morphogenesis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.07.001

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established and one can wonder what could be the consequences of a lack of LYVE-1 down-regulation on collecting vessel maturation. Although still expressed, Prox-1 and VEGFR3 are also observed at lower levels in collecting vessels with the exception of cells constituting lymphatic valves where their expression levels remain high (Fig. 1 and (Norrmen et al., 2009)). A recent study also reports that pre-collectors express lower podoplanin levels than capillaries (Wiig and Swartz, 2012). The functional consequences of this differential protein expression remain to be clarified. It has been reported that the deposition of extracellular matrix (ECM) proteins (collagen IV, laminin α5) constituting the lymphatic basement membrane constitutes an early event in collecting vessel specification (Lutter et al., 2012). It was observed to precede SMC recruitment. However, at late maturation stages, it appears that both endothelial and SMCs are at the source of ECM production (Lutter et al., 2012). The collecting lymphatic vessels are also characterized by the existence of zipper-like junctions between lymphatic endothelial cells that distinguish them from capillaries that exhibit button-like junctions between endothelial cells. During development, zipper to button transformation occurs during capillary specification (Yao et al., 2012). Several studies have decrypted some molecular pathways involved in the remodeling and maturation of the definitive lymphatic vascular network. The transcription factor Foxc2 (forkhead box C2) has been reported to be important for this process. In addition to abnormal SMC coverage of lymphatic capillaries (Petrova et al., 2004), a failure in the down-regulation of VEGFR3, Prox-1 and LYVE-1 has been observed in the collecting vessels of Foxc2-deficient mice (Norrmen et al., 2009). A similar function may also be attributed to angiopoietin-2. Indeed, angiopoietin-2 knockout pups display an abnormal architecture of large lymphatic vessels with a persistent LYVE-1 expression, a lack of valves and an abnormal recruitment of SMC to lymphatic capillaries that correlate with lymphatic dysfunction (Dellinger et al., 2008; Gale et al., 2002). Interestingly, these defects can be rescued by the Tie2 agonist angiopoietin-1. Mice lacking the PDZ domain of ephrinB2 were also found to exhibit SMC coverage of lymphatic capillaries and persistent LYVE-1 expression in the lymphatic vasculature (Makinen et al., 2005). These findings suggest that all these factors, ie. Foxc2, angiopoietin-2 and ephrinB2 are required to prevent SMC coverage of lymphatic capillaries and to remodel the primary lymphatic plexus into a hierarchical functional network. Moreover, they suggest the existence of cross-talks between the mechanisms of action of all these regulators. The Phosphoinositide 3-kinase (PI3K)/Akt pathway may be involved downstream of these regulators since defects in lymphatic vessel remodeling and in collecting vessel maturation have been reported in mice lacking either PI3K regulatory isoforms or Akt1 (Mouta-Bellum et al., 2009) (Zhou et al., 2010). Lymphatic valve formation An essential characteristic of collecting vessels is the presence of intraluminal valves. Lymphatic valves are essential to prevent lymph backflow. They are constituted by a central connective tissue core surrounded by a double layer of endothelial cells (Bazigou et al., 2009). Valve leaflets are anchored at their bases to the vessel wall by collagen and elastin fibers that ensure a proper valve functioning according to the variations of pressure (Davis et al., 2011). Valveforming LECs are defined by a distinct molecular expression profile when compared to capillary or collecting vessel LECs (Fig. 1 and reviewed in (Koltowska et al., 2013), (Yang and Oliver, 2014)). Most of what is known about the process of valve formation comes from studies using genetic mouse mutants focused on mouse mesentery development. Valve formation in the mesentery occurs between E15/E16 and the first postnatal days (Norrmen et al., 2009), (Sabine et al., 2012). Several steps have been characterized during valve morphogenesis (reviewed in (Bazigou and Makinen, 2013), (Koltowska et al., 2013), (Yang and Oliver, 2014)). They are schematically illustrated on Fig. 2.

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The first signs corresponding to the initiation of valve development are the high expression levels of the transcription factors Prox-1 and Foxc2 in discrete clusters of cells that will constitute the future valves. These factors, uniformly expressed in cells of the lymphatic plexus at E15.5, are normally down-regulated during lymphatic vessel remodeling and maturation except in the cells that will constitute the future valves (Norrmen et al., 2009). Prox-1 has been characterized to play a key role for the differentiation along the lymphatic endothelial lineage and to be the master gene for the maintenance of the lymphatic endothelial cell identity (Wigle and Oliver, 1999) (Johnson et al., 2008). However, the significance of its maintained high expression in valve-constituting cells is still not clearly understood. Mice deficient in Foxc2 fail to form lymphatic valves and Foxc2 mutations in humans have been described to lead to the development of lymphedema distichiasis (Petrova et al., 2004). In this pathology, patients display abnormal lymphatic vascular morphogenesis and valve defects (Petrova et al., 2004) (Fang et al., 2000). Foxc2 was found to function in close association with another transcription factor, NFATc1 (Nuclear factor of activated T-cells, cytoplasmic 1) for the control of collecting lymphatic vessel maturation and valve formation (Norrmen et al., 2009). In addition to the role of NFATc1 during the early steps of lymphatic development (Kulkarni et al., 2009), studies using mouse mutants or pharmacological inhibitors have characterized that the calcineurin/NFATc1 signaling pathway is involved in the control of valve morphogenesis downstream of Foxc2 (Norrmen et al., 2009) (Sabine et al., 2012). NFATc1 activation results from its dephosphorylation by the calcium-dependent phosphatase Calcineurin (CNB1). Dephosphorylated NFATc1 can then translocate into the nucleus to regulate target gene transcription. The location of valves is often seen at sites of vessel branching where disturbed lymph flow and/or re-circulation occur (Planas-Paz and Lammert, 2013). This is in accordance with an important role of fluid dynamics and shear stress in the initiation of the valve formation process. This assumption was supported by the findings that lymphatic valve formation correlates with the onset of lymph flow and that non-laminar oscillatory fluid flow can cooperate with Prox-1 and Foxc2 in the induction of crucial genes involved in valve morphogenesis (Sabine et al., 2012). The valve territory is further delimited by a circularization process of valve forming cells. Both CNB1/NFATc1 signaling pathway and Connexins (Cx) 37 and 43 were shown to be required at this stage (Sabine et al., 2012) (Kanady et al., 2011). Connexins are transmembrane proteins that form hemichannels or gap junction channels. Expression of Cx37 has been characterized to be a downstream target of Prox-1 and Foxc2 that further define the identity of valve forming cells (Sabine et al., 2012). This valve cell specification is concomitant with the first signs of extracellular matrix deposition including Laminin α5 and fibronectin EIIIA splice isoform (Bazigou et al., 2009). The valve cell identity was also characterized by the lack of Neuropilin 2 and LYVE-1 expression and particularly high levels of the transcription factor GATA2 (reviewed in (Koltowska et al., 2013)). This factor has been suggested to constitute a regulator of the transcriptional programing of valve development (Koltowska et al., 2013) (Kazenwadel et al., 2012). Indeed, a recent work has established that GATA2 controls the expression of Prox-1 and Foxc2, and that loss of function GATA2 mutations are associated with primary lymphedema (Kazenwadel et al., 2012). Once specified, valve forming cells orientate perpendicular to the longitudinal axis of the vessel and migrate into the vessel lumen forming a ring-like constriction that corresponds with the initiation of leaflet formation (Bazigou et al., 2009) (Sabine et al., 2012) (Tatin et al., 2013). During this process, two components of the planar cell polarity (PCP) signaling pathway were observed to play an important role in the migration and the polarization of the valve forming cells. Core planar cell polarity proteins Cadherin EGF LAG seven-pass G-type receptor 1 (Celsr1) and Van Gogh-like 2 (Vangl2) were found to be specifically implicated in these cell rearrangements (Tatin et al., 2013). Once more, the trigger seems to be the flow that appears to govern

Please cite this article as: Vittet, D., Lymphatic collecting vessel maturation and valve morphogenesis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.07.001

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Fig. 2. Schematic representation of the different stages of lymphatic valve development and of the molecular mechanisms regulating valve morphogenesis. The main steps occurring during lymphatic valve formation and their key regulators are indicated. Valve-forming cells are in dark blue and the valve core matrix and its constituents are in red. The initiation of valve formation is concomitant with the onset of flow that induced Foxc2 expression in cell clusters located at sites of future valves, which in cooperation with Prox1 will regulate the expression of genes and signaling pathways that lead to the establishment of valve territory, vessel constriction and valve leaflet formation (see the text for the detailed role of each molecular player).

the polarized expression of these two proteins, preceding cell reorientation (Tatin et al., 2013) (Shi et al., 2013). Interestingly, a polarization in Cx37 and Cx43 molecular expression profile is observed in cells constituting mature lymphatic valves (reviewed in (Koltowska et al., 2013)), and a gradual expression level for Prox-1 and Foxc2 has been described during the assembly of the valve-forming cells into a ring-like structure on either side of the valve. Cells upstream of the valve express high Prox-1 levels whereas cells downstream of the valve express high Foxc2 levels (Sabine et al., 2012). Valve maturation takes place through the development of functional valve leaflets. This involves both the deposition of a central extracellular matrix core that includes laminin α5, collagen IV and fibronectin EIIIA splice isoform (FN-EIIIA), and the elongation of the leaflets inside the vessel lumen. Valve maturation has been shown to be critically dependent of the cell-matrix adhesion receptor integrin α9 that binds FN-EIIIA. Inactivation of the integrin α9 gene or of its ligand FN-EIIIA in mice results in the formation of a majority of immature ring-like shaped valve and the failure to develop valve leaflets (Bazigou et al., 2009). Interaction of integrin α9 with EMILIN1, an extracellular matrix multi-domain glycoprotein that can also bind integrin α9, has also recently been reported crucial for valve leaflet formation since EMILIN1-/- mice display a similar valve phenotype than integrin α9 or FN- EIIIA-deficient mice (Danussi et al., 2013). A direct function of the axon guidance molecule Semaphorin3A (Sema3A), via the interaction with the Neuropilin-1 (Nrp1) and the PlexinA1 receptors, was also reported on valve leaflet formation (Bouvree et al., 2012). Sema3A, produced by LECs, was postulated to bind to its receptors expressed on valve endothelial cells for generating signals that induce the migration of cells into the vessel lumen to form the valve leaflets (Bouvree et al., 2012) (Jurisic et al., 2012). The remodeling of the lymphatic vasculature of the mouse mesentery with the structure of a mature valve is illustrated on Fig. 3. Genetic studies in mice have also revealed a number of other signaling pathways that play important roles in the regulation of valve formation and maturation. Mutations in the PDZ cytoplasmic domain of the transmembrane ligand ephrinB2 causes valve agenesis (Makinen et al., 2005). Further evidence for a role of this pathway was provided by a study reporting that perturbation of ephrinB2-EphB4 signals disrupts valve formation in corneal lymphatic vessels (Katsuta et al., 2013). Mice lacking angiopoietin-2, the ligand for the endothelial receptor Tie2, also display defects in the maturation of the collecting lymphatic vessels associated with a deficit in intraluminal lymphatic valves (Gale et al., 2002) (Dellinger et al., 2008). However, a direct action on valve

formation, the precise morphogenetic events that are affected by these signaling pathways and the molecular mechanisms involved, remain to be fully characterized. BMP9 (Bone morphogenetic protein 9) is a high affinity ligand for the TGF-β family type 1 receptor Alk1 (Activin receptor-like kinase 1) that has been recently described to be essential for both collecting vessel maturation and valve morphogenesis (Levet et al., 2013). LYVE-1 was established as a target gene for BMP9 and an impairment of LYVE-1 down-regulation in large collecting vessels was observed in the mesentery of BMP9-deficient newborn mice (Levet et al., 2013). Concerning valve formation, BMP9 appears to act at both the initiation and the maturation levels since BMP9-deficient mice display a reduced number of valves, and among those that can have formed a low proportion of mature valves (Levet et al., 2013). Consistent with this assumption, BMP9 was observed to regulate, in primary cultures of LECs, several master genes known to be involved in the initiation of valve formation and/or in the different stages of maturation (Levet et al., 2013). These observations allow one to postulate that BMP9 is an upstream regulator of several genes, including Foxc2, and a crucial factor for lymphatic collecting vessel maturation and valve morphogenesis. In addition, BMP9 constitutes the sole circulating factor involved in these processes that has been identified until now. Vascular wall assembly in lymphatic collecting vessels During their functional maturation, high caliber lymphatic vessels are invested by smooth muscle cells (SMCs). Coverage of pre-collectors remains very sparse and is in some cases absent whereas mature lymphatic collecting vessels are tightly wrapped by SMCs. However, the SMC coverage of collecting vessels remains smaller when compared to the SMC coverage of veins and arteries. The time-course of SMCs recruitment has recently been reported in detail for collecting vessels in mouse ear skin. SMC recruitment was observed to follow the first signs of matrix protein deposition in the basement membrane and to be concomitant with the downregulation of LYVE-1 expression (Lutter et al., 2012). However, a clear correlation between the two events has not been established, and it should be noted that differences are observed depending on the caliber and the tissue localization of the collecting vessels. Indeed, the large collecting vessels of the mesentery exhibit a more elaborate and continuous SMC coverage than those of the ear, and LYVE-1 down-regulation was found to occur once SMC coverage has been achieved (Lutter et al., 2012).

Please cite this article as: Vittet, D., Lymphatic collecting vessel maturation and valve morphogenesis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.07.001

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Fig. 3. lymphatic vascular remodeling and valve morphology in mouse mesentery collecting vessels. (A) Lymphatic collecting vessel maturation and the morphogenetic process of valve development in the mouse mesentery occurs between E15/E16 and early postnatal days. Mesenteric vessels stained for Prox1 (green) and CD31 (red) are illustrated. LV, lymphatic vessel; A, artery; V, vein. Arrowsheads pointed to valve location where a high Prox1 immunoreactivity is observed in the nuclei of valve-constituting cells. (B) Structure of a mature lymphatic valve. Representative views of bi-leaflet V-shaped mature valves visualized after Prox1, CD31, Laminin α5 and αSMA staining. Lymphatic valve cells overexpress Prox1 and display high CD31 immunoreactivity. Laminin α5 staining allows the visualization of the central connective tissue core of the valve. The valve territory is devoid of smooth muscle coverage as indicated by the lack of αSMA positive cells at the valve location.

The molecular mechanisms regulating SMC coverage of collecting vessels have been poorly investigated (Fig. 4). As for blood vessels, the vascular smooth muscle cell chemoattractant PDGF-B seems to be involved. In lymphatic endothelial cells, PDGF-B expression has been described to be under the control of Foxc2 (Petrova et al., 2004). The extracellular matrix glycoprotein Reelin has been recently identified as a regulator of SMCs recruitment in ear skin collecting vessels and a proper SMC-LEC interaction appears to play a critical role in the control of collecting vessel morphogenesis and function (Lutter et al., 2012). It was demonstrated that the contact of SMC with LEC induces the secretion of Reelin from LECs, which in an autocrine manner, by a noncanonical signaling pathway remaining to be elucidated, stimulates the expression of MCP1 (monocyte chemotactic protein 1) in LECs, and further enhances SMC recruitment to the vessel. Moreover, a functional impairment of the draining efficiency is observed in Reelindeficient mice (Lutter et al., 2012). Nevertheless, this process does not appear to be involved in collecting vessels of the mesentery, thus pointing to the existence of different mechanisms regulating collecting vessel maturation depending on the tissue environment (Lutter et al.,

2012). Moreover, the molecular actors driving the initial contact between SMCs and LECs that trigger the full cascade of the SMC recruitment process have not been identified. Valve areas are mostly free of SMC coverage probably in order to not interfere with their functioning. Indeed, valve gating is assumed to be passively governed by variations in intraluminal pressure gradients although several observations also mentioned the influence of mechanical factors related to vessel distension (Davis et al., 2011). Analysis of the muscle cell investiture of mesenteric collecting vessels revealed the presence of a majority of circularly-oriented muscle cells covering the lymphangions and that some longitudinally-oriented muscle cells cross-connect lymphangions over valve areas (Bridenbaugh et al., 2013). As suggested, these cells may be involved in the electrical coupling between two successive lymphangions, ensuring the propagation of the contractile activity, and may affect valve gating (Bridenbaugh et al., 2013). The Semaphorin3A (Sema3A)/Neuropilin-1 (NRP1) signaling pathway is the sole mechanism identified until now that maintains the valve area free of SMC coverage (Jurisic et al., 2012), (Bouvree et al., 2012). In these studies, the binding of Sema3A (produced by valve-

Please cite this article as: Vittet, D., Lymphatic collecting vessel maturation and valve morphogenesis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.07.001

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valve formation in tissues of different location. The heterogeneity of the lymphatic vessels according to the tissue environment remains mostly elusive until now. Finally, it appears important to better characterize what are the consequences of collecting vessels dilation and/or of valve deficiency in lymphatic-associated pathologies and in tumor metastasis. Indeed, it seems reasonable to speculate that dysfunction of lymphatic vasculature could be responsible for variations in the efficiency of the immune response or in the metastatic spreading of certain tumors. Acknowledgments The author thanks Dr. Sabine Bailly and Dr. Jean-Jacques Feige for critical reading of the manuscript and Dr. Lydia Sorokin (Münster University, Germany) for generously providing the laminin α5 antibody. This research work was supported by Inserm (U1036), CEA (iRTSV/ BCI), UJF, Fondation pour la recherche sur le cancer (grant n° PJA 20131200252) and the comités départementaux de la Loire et de l’Isère de la ligue contre le cancer. Fig. 4. Model for lymphatic collecting vessel wall asssembly. The coverage of lymphatic collecting vessels by smooth muscle cells (SMC) is demonstrated in the ear to be controlled by Reelin produced by lymphatic endothelial cells (LECs) that acts in an autocrine manner to induce the secretion of the SMC recruitment factor Monocyte Chemotactic Protein 1 (MCP1). Valve areas are maintained free of SMCs coverage following the repulsive signal between Semaphorin3A (Sema3A) secreted by lymphatic valve-forming cells and Neuropilin1 (NRP1) expressed on SMCs.

forming endothelial cells) to NRP1 receptor expressed by SMCs, was shown to repulse SMCs from valve areas. Concluding remarks and future research directions Several processes occurring during the development and the maturation of lymphatic collecting vessels appear to be linked. Defects in collecting vessel specification result in the failure to form valves. Despite great advances obtained during the past ten years in the understanding of the mechanisms of lymphatic vascular remodeling, patterning and maturation, many questions remain unsolved. Although several important regulators of lymphatic vessel maturation and valve formation have been identified, there probably exist additional factors and signaling pathways not identified yet, that are likely to be involved in the regulation of these processes. In particular, it would be essential to characterize all the inducers of the initiation of valve formation. The importance of the exposure to flow has been established and lymphatic endothelial cell mechanosensing is emerging as a critical regulator of lymphangiogenesis and valve formation. However, the mechanosensor(s) that sense the flow and the molecular factors involved in the regulation of Prox1 and Foxc2 expressions have not been fully identified. New valve formation has not been described until now in adult mature lymphatic vessels (Bazigou et al., 2009) (Norrmen et al., 2009). It would be interesting to check whether there is a possibility to form new lymphatic valves in established adult collecting vessels and to better analyze the mechanisms of valve maintenance in order to prevent their regression. This may be of importance in the perspective of lymphatic draining improvement in the case of lymphedema. In this context, it appears also necessary to get better insight in the mechanisms regulating lymphatic collecting vessel contractile activity. Are they similar to those regulating the blood vessel vascular tone? There are different requirements for SMC recruitment depending on the caliber of the lymphatic vessel. What is the trigger of SMC recruitment? Several differences have also been reported in the kinetics of lymphatic vessel maturation and valve formation between the mesentery and the ear skin. One may wonder if the regulatory mechanisms involved may be different. Then, one of the future challenges will be to elucidate the precise contributions and roles of the identified factors and signaling pathways involved in collecting vessel maturation and

References Bazigou, E., Makinen, T., 2013. Flow control in our vessels: vascular valves make sure there is no way back. Cell Mol. Life Sci. 70, 1055–1066. Bazigou, E., Xie, S., Chen, C., Weston, A., Miura, N., Sorokin, L., Adams, R., Muro, A.F., Sheppard, D., Makinen, T., 2009. Integrin-alpha9 is required for fibronectin matrix assembly during lymphatic valve morphogenesis. Dev. Cell 17, 175–186. Bouvree, K., Brunet, I., Del Toro, R., Gordon, E., Prahst, C., Cristofaro, B., Mathivet, T., Xu, Y., Soueid, J., Fortuna, V., Miura, N., Aigrot, M.S., Maden, C.H., Ruhrberg, C., Thomas, J.L., Eichmann, A., 2012. Semaphorin3A, Neuropilin-1, and PlexinA1 are required for lymphatic valve formation. Circ. Res. 111, 437–445. Bridenbaugh, E.A., Nizamutdinova, I.T., Jupiter, D., Nagai, T., Thangaswamy, S., Chatterjee, V., Gashev, A.A., 2013. Lymphatic muscle cells in rat mesenteric lymphatic vessels of various ages. Lymphat. Res. Biol. 11, 35–42. Danussi, C., Del Bel Belluz, L., Pivetta, E., Modica, T.M., Muro, A., Wassermann, B., Doliana, R., Sabatelli, P., Colombatti, A., Spessotto, P., 2013. EMILIN1/alpha9beta1 integrin interaction is crucial in lymphatic valve formation and maintenance. Mol. Cell. Biol. 33, 4381–4394. Davis, M.J., Rahbar, E., Gashev, A.A., Zawieja, D.C., Moore Jr., J.E., 2011. Determinants of valve gating in collecting lymphatic vessels from rat mesentery. Am. J. Physiol. Heart Circ. Physiol. 301, H48–H60. Dellinger, M., Hunter, R., Bernas, M., Gale, N., Yancopoulos, G., Erickson, R., Witte, M., 2008. Defective remodeling and maturation of the lymphatic vasculature in Angiopoietin-2 deficient mice. Dev. Biol. 319, 309–320. Fang, J., Dagenais, S.L., Erickson, R.P., Arlt, M.F., Glynn, M.W., Gorski, J.L., Seaver, L.H., Glover, T.W., 2000. Mutations in FOXC2 (MFH-1), a forkhead family transcription factor, are responsible for the hereditary lymphedema-distichiasis syndrome. Am. J. Hum. Genet. 67, 1382–1388. Francois, M., Harvey, N.L., Hogan, B.M., 2011. The transcriptional control of lymphatic vascular development. Physiology (Bethesda) 26, 146–155. Gale, N.W., Thurston, G., Hackett, S.F., Renard, R., Wang, Q., McClain, J., Martin, C., Witte, C., Witte, M.H., Jackson, D., Suri, C., Campochiaro, P.A., Wiegand, S.J., Yancopoulos, G.D., 2002. Angiopoietin-2 is required for postnatal angiogenesis and lymphatic patterning, and only the latter role is rescued by Angiopoietin-1. Dev. Cell 3, 411–423. Gale, N.W., Prevo, R., Espinosa, J., Ferguson, D.J., Dominguez, M.G., Yancopoulos, G.D., Thurston, G., Jackson, D.G., 2007. Normal lymphatic development and function in mice deficient for the lymphatic hyaluronan receptor LYVE-1. Mol. Cell. Biol. 27, 595–604. Hagerling, R., Pollmann, C., Andreas, M., Schmidt, C., Nurmi, H., Adams, R.H., Alitalo, K., Andresen, V., Schulte-Merker, S., Kiefer, F., 2013. A novel multistep mechanism for initial lymphangiogenesis in mouse embryos based on ultramicroscopy. EMBO J. 32, 629–644. Johnson, N.C., Dillard, M.E., Baluk, P., McDonald, D.M., Harvey, N.L., Frase, S.L., Oliver, G., 2008. Lymphatic endothelial cell identity is reversible and its maintenance requires Prox1 activity. Genes Dev. 22, 3282–3291. Jurisic, G., Maby-El Hajjami, H., Karaman, S., Ochsenbein, A.M., Alitalo, A., Siddiqui, S.S., Ochoa Pereira, C., Petrova, T.V., Detmar, M., 2012. An unexpected role of semaphorin3a-neuropilin-1 signaling in lymphatic vessel maturation and valve formation. Circ. Res. 111, 426–436. Kanady, J.D., Dellinger, M.T., Munger, S.J., Witte, M.H., Simon, A.M., 2011. Connexin37 and Connexin43 deficiencies in mice disrupt lymphatic valve development and result in lymphatic disorders including lymphedema and chylothorax. Dev. Biol. 354, 253–266. Karkkainen, M.J., Haiko, P., Sainio, K., Partanen, J., Taipale, J., Petrova, T.V., Jeltsch, M., Jackson, D.G., Talikka, M., Rauvala, H., Betsholtz, C., Alitalo, K., 2004. Vascular endothelial growth factor C is required for sprouting of the first lymphatic vessels from embryonic veins. Nat. Immunol. 5, 74–80. Katsuta, H., Fukushima, Y., Maruyama, K., Hirashima, M., Nishida, K., Nishikawa, S., Uemura, A., 2013. EphrinB2-EphB4 signals regulate formation and maintenance of

Please cite this article as: Vittet, D., Lymphatic collecting vessel maturation and valve morphogenesis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.07.001

D. Vittet / Microvascular Research xxx (2014) xxx–xxx funnel-shaped valves in corneal lymphatic capillaries. Invest. Ophthalmol. Vis. Sci. 54, 4102–4108. Kazenwadel, J., Secker, G.A., Liu, Y.J., Rosenfeld, J.A., Wildin, R.S., Cuellar-Rodriguez, J., Hsu, A.P., Dyack, S., Fernandez, C.V., Chong, C.E., Babic, M., Bardy, P.G., Shimamura, A., Zhang, M.Y., Walsh, T., Holland, S.M., Hickstein, D.D., Horwitz, M.S., Hahn, C.N., Scott, H.S., Harvey, N.L., 2012. Loss-of-function germline GATA2 mutations in patients with MDS/AML or MonoMAC syndrome and primary lymphedema reveal a key role for GATA2 in the lymphatic vasculature. Blood 119, 1283–1291. Koltowska, K., Betterman, K.L., Harvey, N.L., Hogan, B.M., 2013. Getting out and about: the emergence and morphogenesis of the vertebrate lymphatic vasculature. Development 140, 1857–1870. Kulkarni, R.M., Greenberg, J.M., Akeson, A.L., 2009. NFATc1 regulates lymphatic endothelial development. Mech. Dev. 126, 350–365. Levet, S., Ciais, D., Merdzhanova, G., Mallet, C., Zimmers, T.A., Lee, S.J., Navarro, F.P., Texier, I., Feige, J.J., Bailly, S., Vittet, D., 2013. Bone morphogenetic protein 9 (BMP9) controls lymphatic vessel maturation and valve formation. Blood 122, 598–607. Lutter, S., Xie, S., Tatin, F., Makinen, T., 2012. Smooth muscle-endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation. J. Cell Biol. 197, 837–849. Makinen, T., Adams, R.H., Bailey, J., Lu, Q., Ziemiecki, A., Alitalo, K., Klein, R., Wilkinson, G.A., 2005. PDZ interaction site in ephrinB2 is required for the remodeling of lymphatic vasculature. Genes Dev. 19, 397–410. Margaris, K.N., Black, R.A., 2012. Modelling the lymphatic system: challenges and opportunities. J. R. Soc. Interface 9, 601–612. Martinez-Corral, I., Makinen, T., 2013. Regulation of lymphatic vascular morphogenesis: Implications for pathological (tumor) lymphangiogenesis. Exp. Cell Res. 319, 1618–1625. Mouta-Bellum, C., Kirov, A., Miceli-Libby, L., Mancini, M.L., Petrova, T.V., Liaw, L., Prudovsky, I., Thorpe, P.E., Miura, N., Cantley, L.C., Alitalo, K., Fruman, D.A., Vary, C.P., 2009. Organspecific lymphangiectasia, arrested lymphatic sprouting, and maturation defects resulting from gene-targeting of the PI3K regulatory isoforms p85alpha, p55alpha, and p50alpha. Dev. Dyn. 238, 2670–2679. Norrmen, C., Ivanov, K.I., Cheng, J., Zangger, N., Delorenzi, M., Jaquet, M., Miura, N., Puolakkainen, P., Horsley, V., Hu, J., Augustin, H.G., Yla-Herttuala, S., Alitalo, K., Petrova, T.V., 2009. FOXC2 controls formation and maturation of lymphatic collecting vessels through cooperation with NFATc1. J. Cell Biol. 185, 439–457.

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Petrova, T.V., Karpanen, T., Norrmen, C., Mellor, R., Tamakoshi, T., Finegold, D., Ferrell, R., Kerjaschki, D., Mortimer, P., Yla-Herttuala, S., Miura, N., Alitalo, K., 2004. Defective valves and abnormal mural cell recruitment underlie lymphatic vascular failure in lymphedema distichiasis. Nat. Med. 10, 974–981. Planas-Paz, L., Lammert, E., 2013. Mechanical forces in lymphatic vascular development and disease. Cell Mol. Life Sci. 70, 4341–4354. Sabine, A., Agalarov, Y., Maby-El Hajjami, H., Jaquet, M., Hagerling, R., Pollmann, C., Bebber, D., Pfenniger, A., Miura, N., Dormond, O., Calmes, J.M., Adams, R.H., Makinen, T., Kiefer, F., Kwak, B.R., Petrova, T.V., 2012. Mechanotransduction, PROX1, and FOXC2 cooperate to control connexin37 and calcineurin during lymphatic-valve formation. Dev. Cell 22, 430–445. Schulte-Merker, S., Sabine, A., Petrova, T.V., 2011. Lymphatic vascular morphogenesis in development, physiology, and disease. J. Cell Biol. 193, 607–618. Shi, D., Fujimori, T., Uemura, T., 2013. Atypical cadherin negotiates a turn. Dev. Cell 26, 1–2. Tammela, T., Alitalo, K., 2010. Lymphangiogenesis: Molecular mechanisms and future promise. Cell 140, 460–476. Tatin, F., Taddei, A., Weston, A., Fuchs, E., Devenport, D., Tissir, F., Makinen, T., 2013. Planar cell polarity protein Celsr1 regulates endothelial adherens junctions and directed cell rearrangements during valve morphogenesis. Dev. Cell 26, 31–44. Wigle, J.T., Oliver, G., 1999. Prox1 function is required for the development of the murine lymphatic system. Cell 98, 769–778. Wiig, H., Swartz, M.A., 2012. Interstitial fluid and lymph formation and transport: physiological regulation and roles in inflammation and cancer. Physiol. Rev. 92, 1005–1060. Yang, Y., Oliver, G., 2014. Development of the mammalian lymphatic vasculature. J. Clin. Invest. 124, 888–897. Yang, Y., Garcia-Verdugo, J.M., Soriano-Navarro, M., Srinivasan, R.S., Scallan, J.P., Singh, M. K., Epstein, J.A., Oliver, G., 2012. Lymphatic endothelial progenitors bud from the cardinal vein and intersomitic vessels in mammalian embryos. Blood 120, 2340–2348. Yao, L.C., Baluk, P., Srinivasan, R.S., Oliver, G., McDonald, D.M., 2012. Plasticity of buttonlike junctions in the endothelium of airway lymphatics in development and inflammation. Am. J. Pathol. 180, 2561–2575. Zhou, F., Chang, Z., Zhang, L., Hong, Y.K., Shen, B., Wang, B., Zhang, F., Lu, G., Tvorogov, D., Alitalo, K., Hemmings, B.A., Yang, Z., He, Y., 2010. Akt/Protein kinase B is required for lymphatic network formation, remodeling, and valve development. Am. J. Pathol. 177, 2124–2133.

Please cite this article as: Vittet, D., Lymphatic collecting vessel maturation and valve morphogenesis, Microvasc. Res. (2014), http://dx.doi.org/ 10.1016/j.mvr.2014.07.001