VE-Cadherin: A Critical Sticking Point for Lymphatic System Maintenance

VE-cadherin plays a multitude of roles in endothelial cells, including the vital function of cell-cell adhesion and interaction as well as transmembrane signal transduction. These adhesion molecules provide the foundational function of regulating vessel permeability. Vascular permeability is intimately linked with immune cell transportation, solute concentration gradient establishment, and fluid homeostasis. With booming interest in the role of lymphatic vessels in normal and pathologic states, the importance of lymphatic endothelial cell (LEC) interaction in vessel development, maintenance, and function has come into focus. Recent ex vivo models of lymphatic vessel permeability have demonstrated a significant increase in permeability to both small and large solutes when vessels are exposed to functional VE-cadherin blockade¹. The mechanotransducive role of constitutively bound VE-cadherin to VEGFR2 and VEGFR3 has been previously demonstrated in blood endothelial cells (BECs)², and more recently in the formation and maintenance of lymphatic valves³, further bolstering the evidence of the critical role VE-cadherin plays in the realm of lymphatics.

Additionally, VE-cadherin has been demonstrated to have organ-specific roles in lymphatic vessels, namely in the development and maintenance of distinct lymphatic beds like the dermal lymphatics, lacteals, and mesentery, and the development of the intraluminal lymphatic valves⁴. However, to date, the involvement of VE-cadherin in cardiac lymphatic system development and lymphangiogenic response in disease states has not been significantly explored. The essential nature of lymphangiogenesis in diseases such as myocardial infarction has been demonstrated through the impairment of post-myocardial infarction cardiac function in dysfunctional lymphatic murine models, though recent evidence showing maintenance of baseline cardiac function after injury despite disrupted lymphangiogenesis via VEGFR3 deletion has come to light⁵.

In their current study, Harris et al.⁶ provide strong mechanistic signaling data that elucidates a key role for VE-cadherin in establishing a signaling node of powerful lymphangiocrine factors, VEGF-C and adrenomedullin. The authors further defined the relationship between VE-cadherin and VEGFR3, demonstrating that disruption of VE-cadherin expression leads to significant hinderance of the canonical activation of VEGFR3 by VEGF-C. They also identify a novel mechanism for VEGFR3 transactivation by the cardioprotective and lymphangiogenic peptide adrenomedullin (AM).

Canonical VEGFR3 activation is triggered by ligand binding, typically VEGF-C, leading to autophosphorylation and downstream signaling, including ERK or AKT phosphorylation. However, VEGFR3 is receptive to c-Src-mediated transactivation on a subset of residues that are not activated by ligand-mediated autophosphorylation⁷. Utilizing a proximity ligation assay (PLA) with VEGFR3 and pan-phosphotyrosine antibodies in cultured LECs, Harris et al.⁶ demonstrate significant VEGFR3 phosphorylation by AM when canonical VEGF-C activation is blocked, via the small molecule inhibitor sunitinib. They also show AM transactivation is mediated through c-Src, like other established methods of VEGFR3 transactivation and reminiscent of the mechanism by which AM transactivates VEGFR2 in blood endothelial cells. Further, the authors show that VEGFR3 activation by VEGF-C or transactivation by AM is abrogated by loss of VE-cadherin. This finding is consistent with the established role of VE-cadherin in assembling endothelial flow sensing complexes at cell-cell junctions, namely VEGFR2 and VEGFR3².

Of note, both AM/CLR and VEGFC/VEGFR3 signaling are required for active lymphangiogenesis during development and are key regulators of LEC migration and barrier function ^{8, 9}. With their co-expression in the lymphatic endothelium and their parallel activities towards common goals, it is perhaps not surprising to observe such a striking impact of VE-cadherin loss on the cardiac lymphatic network. The clinical implications of these converged pathways is most likely utilized during the cardiac repair response after myocardial infarction (MI), a time when plasma levels of AM are dramatically upregulated in ischemic injury¹⁰ and therapeutic stimulation of VEGF-C signaling has shown robust increases in cardiac lymphangiogenesis^11–13.

Harris et al.⁶ additionally provide substantial conceptual advances to our prior knowledge by evaluating the temporal effects of VE-cadherin loss on cardiac lymphatic anatomy, through deletion of the gene during embryogenesis, early postnatal period, and adulthood. An interesting finding is that deletion of VE-cadherin is temporally-sensitive—the most prominent effects were observed in the postnatal period and in the temporal regression of lymphatics from adult-deletion animals—indicating that VE-cadherin imparts different cellular effects on lymphatics depending on the stage in which its expression is deleted. It is additionally poignant to note that, despite the anatomic regression of cardiac lymphatic networks seen from VE-cadherin deletion in either postnatal or adult mice, cardiac function is preserved on echocardiographic evaluation and without significant increase in left ventricular (LV) mass compared to control.

The preserved functional cardiac phenotype stands in stark contrast to the resultant nutritional malabsorption and chylous ascites from disrupted gastrointestinal lymphatics in postnatal VE-cadherin deletion. This lends evidence to differential functional phenotypic expressions seen with the deletion or impairment of VE-cadherin based anatomical location of lymphatic beds. Here, Harris et al.⁶ demonstrated this beautifully with parallel functional assays of both dermal and cardiac lymphatics. A multi-point approach was employed to assess the function of dermal lymphatics in Cdh5^LEC-KO mice. They tested the ability of dermal lymphatics to resolve edema following injection of an inflammatory stimulus and found a significant increase in initial paw edema in the Cdh5^LEC-KO mice coupled with an inability to resolve this edema over time. Parallel assays using tail microlymphography also revealed a profound phenotype in Cdh5^LEC-KO mice, with failure of lymphatic capillary dye uptake. High resolution 3-dimensional light sheet microscopy of tail lymphatics confirmed their structural integrity, despite the marked loss of function.

In a parallel series of elegant approaches, the team also used fluorescent quantum dots to perform cardiac lymphangiography to directly assess cardiac lymphatic drainage in anesthetized Cdh5^LEC-KO mice. Cardiac lymphatics devoid of VE-cadherin were significantly deficient in both dye uptake and transport, with apparent extravasation of contrast proximal to the point of injection.

This novel genetic mouse model that displays dysfunctional cardiac lymphatics provided an ideal setting for the evaluation of whether the absence or dysfunction of cardiac lymphatics could influence the lymphangiogenic response after myocardial injury. Perhaps expectedly, Cdh5^LEC-KO mice failed to respond to lymphangiocrine signals induced after ischemic injury, as very little lymphatics were detected in post-MI cardiac histological sections in comparison to wild type mice. However, one of the most surprising findings was that cardiac lymphatics appeared to be dispensable for preserving basal cardiac function before and after injury. This was unexpected because several studies have recently shown that active lymphangiogenesis after myocardial infarction (either by VEGFC supplementation or adrenomedullin over-expression) is beneficial to heart recovery^{11, 14, 15}. On the other hand, the current study clearly demonstrates that loss and/or dysfunction of cardiac lymphatics does not negatively affect heart recovery—a finding which has also been very recently described in mice lacking VEGFR3 signaling ⁵. Overall, these studies indicate that expression of VE-cadherin is required for the normal function and maintenance of the cardiac lymphatic vessel network, but that cardiac lymphatic absence or dysfunction does not significantly hinder functional cardiac recovery after myocardial infarction.

In summary, Harris et al.⁶ provide a tour de force in cardiac lymphatics. The mechanistic description demonstrates the necessity of VE-cadherin in both canonical VEGFR3 signaling and non-canonical adrenomedullin signaling at vital time points in cardiac lymphatic development. With expanding knowledge of the differential impact of deficits in adhesion molecules by anatomic location, this study serves as a comprehensive foray into the role of VE-cadherin in cardiac lymphatic maintenance, all the while furthering the discussion of the putative role of the lymphatic system in cardiac fluid homeostasis and post-injury cardiac recovery.

Figure.

VE-cadherin plays an essential role in vascular endothelial growth factor receptor 3 (VEGFR3) signaling through maintenance of membrane localization of the receptor, allowing for VEGFR3 autophosphorylation upon activation by ligand VEGF-C. Additional non-canonical transactivation of VEGFR3 signaling via the adrenomedullin (AM)/calcitonin receptor-like receptor (CLR) contributes to development and maintenance lymphatic endothelial cell (LEC) identity. In the absence of VE-cadherin, VEGFR3 is internalized and signaling via canonical or non-canonical pathways is inhibited. The effect of this is both temporally and anatomically differential, resulting in a number of lymphatic network and functional changes.