The clinical problems that arise from venous valve incompetence highlight the importance of valves in ensuring unidirectional flow and efficient return of blood, especially from the lower extremities. However, the mechanisms that regulate venous valve development have not been described. In the present study, we used genetic reporter strains, SEM, and whole-mount immunofluorescence to characterize the morphology and development of murine venous valves. Valve development was initiated by endothelial cell reorientation followed by ingrowth to form a circumferential shelf, then development of first one and then a second commissure. Notably, the first anatomical description of venous valve development by Kampmeier in the early 20th century accurately captured the basic morphogenic events (16
). The process of valve development described in the present study is also comparable to our previous description of lymphatic valve formation (3
), with the exception that we did not previously describe the single commissure stage.
SEM analyses of mouse and human venous valves revealed morphological differences between the endothelial cells in different parts of the vessel. We found that the cells located upstream of the valve were elongated and aligned in the direction of flow, whereas the cells on the leaflet and downstream of the valve had a rounded morphology, as previously described in venules (17
). A similar arrangement of cells was observed in lymphatic valves. Such differences likely arise from differential exposure to fluid shear stress, which suggests that flow patterns are involved in modulating endothelial cell phenotypes within the valve. In agreement with this suggestion, endothelial cells of lymphatic capillaries exposed to low shear stress levels showed no alignment, whereas cells of the thoracic duct, the largest lymphatic vessel, were aligned in the direction of flow. Previous studies have shown the involvement of shear stress generated by flowing blood in patterning the developing vasculature by, for example, regulating branching morphogenesis and asymmetric remodeling of the aortic arches (18
). At the cellular level, flow-induced endothelial responses include instantaneous ion fluxes as well as signaling cascades that lead to changes in gene expression and actin cytoskeleton (21
). Interestingly, we observed a boundary in β-gal staining in the Tie2lacZ
mice and induction of Efnb2GFP
reporter activity concomitant with the early stages of valve formation. This could be related to the flow patterns around the developing valve that may be involved in establishing its gene expression profile and structure. Exposure of cultured cells to shear stress in vitro leads to upregulation and phosphorylation of Tie2 (22
). In addition, expression of angiopoietin-2, an antagonistic Tie2 ligand, is downregulated under shear stress (23
), which suggests that Tie2-mediated signaling may be involved in regulating endothelial cell responses to flow.
Gene expression analyses revealed that several genes that are highly expressed in lymphatic valves were also present in venous valves; these included Itga9
, and Vegfr3
. Notably, these 3 genes have previously been characterized as lymphatic-specific markers and critical regulators of lymphangiogenesis (3
), with the exception of Vegfr3, which is also expressed in fenestrated and newly formed blood vessels and regulates angiogenesis (25
). In particular, expression of Prox1 in venous valves is surprising, given its well-established role as the master regulator of lymphatic endothelial cell fate (24
). Unexpectedly, specific expression of ephrin-B2, a key arterial and lymphatic marker (4
), was also found in the venous valves. In fact, induction of Efnb2
expression by venous endothelial cells represented one of the earliest stages in valve development. These expression data suggest that valve endothelial cells have a unique identity that is likely attributed to the function of the valve rather than to the type of vessel in which it develops. Furthermore, our findings suggest that venous endothelial cells exhibit plasticity in their ability to take on a different phenotypic identity, thus challenging the common view that endothelial cells in different types of vessels represent distinct terminally differentiated cell lineages whose identity is strictly genetically determined. The importance of environmental cues in regulating endothelial cell identities was previously highlighted by the observation that arterial and venous identities could be altered by changing the flow conditions in the vessels (19
Generation of a mouse line expressing tamoxifen-inducible Cre recombinase under the control of the Prox1
promoter allowed us to target endothelial cells of both developing and mature venous valves. Integrin-α9 and ephrin-B2, the previously identified regulators of lymphatic valve development, were required for venous valve morphogenesis. We believe our study establishes these 2 genes as the first-described regulators of this process. Integrin-α9 and one of its ligands, Fn-EIIIA, are required for the assembly of fibronectin and organization of the extracellular matrix core of the lymphatic valve leaflets (3
), but the cellular function of ephrin-B2 in valve formation remains unknown. As observed in lymphatic valves, Itga9
- as well as Fn-EIIIA
–deficient venous valves had short leaflets, suggestive of similar function. Continuous integrin-α9 and ephrin-B2 signaling was also found to be required for the maintenance of valves, but whereas integrin-α9 depletion in mature venous valves led to their regression, the morphology of lymphatic valves was not affected during the same time period. This could be because there is a higher requirement for repair of venous valves, which are exposed to harsher hemodynamic conditions compared with valves in lymphatic vessels. These results suggest that valve maintenance requires continuous synthesis and assembly of the extracellular matrix core of the leaflet and that any defects in these processes can lead to degeneration of venous and lymphatic valves.
In summary, our data identified what we believe to be the first molecular regulators of venous valves and highlighted involvement of common morphogenetic processes and signaling pathways in controlling valve formation in veins and lymphatic vessels.