Unique functions for Notch4 in murine embryonic lymphangiogenesis.

In mice, embryonic dermal lymphatic development is well understood and used to study gene functions in lymphangiogenesis. Notch signaling is an evolutionarily conserved pathway that modulates cell fate decisions, which has been shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. Actively growing embryonic dermal lymphatics expressed NOTCH1, NOTCH4, and DLL4 which correlated with Notch activity. In lymphatic endothelial cells (LECs), DLL4 activation of Notch induced a subset of Notch effectors and lymphatic genes, which were distinctly regulated by Notch1 and Notch4 activation. Treatment of LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts and differentially and temporally regulated the expression of Notch1 and Hes/Hey genes. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts which correlated with reduced vessel caliber in the maturing lymphatic plexus at E14.5 and reduced branching at E16.5. Activation of Notch4 suppressed LEC migration in a wounding assay significantly more than Notch1, suggesting a dominant role for Notch4 in regulating LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ LECs led to increased lymphatic density consistent with an increase in LEC proliferation, described for the loss of LEC Notch1. Moreover, loss of Notch4 did not affect LEC canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.

Lymphatic Endothelial Cell Defects in Congenital Cardiac Patients With Postoperative Chylothorax.

OBJECTIVES: Chylothorax following cardiac surgery for congenital cardiac anomalies is a complication associated with severe morbidities and mortality. We hypothesize that there are intrinsic defects in the lymphatics of congenital cardiac patients. METHODS: Postsurgical chylothorax lymphatic endothelial cells (pcLECs) (n = 10) were isolated from the chylous fluid from congenital cardiac defect patients, and characterized by fluorescent-activated cell sorting, immunofluorescent staining, and quantitative RT-PCR. Results were compared to normal human dermal lymphatic endothelial cells (HdLECs). pcLECs (n = 3) and HdLECs were xenografted into immunocompromised mice. Implants and postoperative chylothorax patient's pulmonary tissues were characterized by immunostaining for lymphatic endothelial proteins. RESULTS: pcLECs expressed endothelial markers VECADHERIN, CD31, VEGFR2, lymphatic endothelial markers PROX1, podoplanin, VEGFR3, and progenitor endothelial markers CD90 and CD146. However, pcLECs had key differences relative to HdLECs, including altered expression and mislocalization of junctional proteins (VECADHERIN and CD31), and essential endothelial proteins, VEGFR2, VEGFR3, and PROX1. When xenografted in mice, pcLECs formed dilated lymphatic channels with poor cell-cell association. Similar to congenital lymphatic anomalies, the pulmonary lymphatics were dilated in a patient who developed postoperative chylothorax after cardiac surgery. CONCLUSIONS: Recent studies have shown that some postoperative chylothoraces in congenital cardiac anomalies are associated with anatomical lymphatic defects. We found that pcLECs have defects in expression and localization of proteins necessary to maintain lymphatic specification and function. This pcLEC phenotype is similar to that observed in lymphatic endothelial cells from congenital lymphatic anomalies. Co-existence of lymphatic anomalies should be considered as a feature of congenital cardiac anomalies.