YAP and TAZ maintain PROX1 expression in the developing lymphatic and lymphovenous valves in response to VEGF-C signaling.
ABSTRACT: Lymphatic vasculature is an integral part of digestive, immune and circulatory systems. The homeobox transcription factor PROX1 is necessary for the development of lymphatic vessels, lymphatic valves (LVs) and lymphovenous valves (LVVs). We and others previously reported a feedback loop between PROX1 and vascular endothelial growth factor-C (VEGF-C) signaling. PROX1 promotes the expression of the VEGF-C receptor VEGFR3 in lymphatic endothelial cells (LECs). In turn, VEGF-C signaling maintains PROX1 expression in LECs. However, the mechanisms of PROX1/VEGF-C feedback loop remain poorly understood. Whether VEGF-C signaling is necessary for LV and LVV development is also unknown. Here, we report for the first time that VEGF-C signaling is necessary for valve morphogenesis. We have also discovered that the transcriptional co-activators YAP and TAZ are required to maintain PROX1 expression in LVs and LVVs in response to VEGF-C signaling. Deletion of Yap and Taz in the lymphatic vasculature of mouse embryos did not affect the formation of LVs or LVVs, but resulted in the degeneration of these structures. Our results have identified VEGF-C, YAP and TAZ as a crucial molecular pathway in valve development.
Project description:During embryonic lymphatic development, a homeobox transcription factor Prox1 plays important roles in sprouting and migration of a subpopulation of blood vessel endothelial cells (BECs) toward VEGF-C-expressing cells. However, effects of Prox1 on endothelial cellular behavior remain to be elucidated. Here, we show that Prox1, via induction of integrin alpha9 expression, inhibits sheet formation and stimulates motility of endothelial cells. Prox1-expressing BECs preferentially migrated toward VEGF-C via up-regulation of the expression of integrin alpha9 and VEGF receptor 3 (VEGFR3). In mouse embryos, expression of VEGFR3 and integrin alpha9 is increased in Prox1-expressing lymphatic endothelial cells (LECs) compared with BECs. Knockdown of Prox1 expression in human LECs led to decrease in the expression of integrin alpha9 and VEGFR3, resulting in the decreased chemotaxes toward VEGF-C. These findings suggest that Prox1 plays important roles in conferring and maintaining the characteristics of LECs by modulating multiple signaling cascades and that integrin alpha9 may function as a key regulator of lymphangiogenesis acting downstream of Prox1.
Project description:Lymphatic valves (LVs) are cusped luminal structures that permit the movement of lymph in only one direction and are therefore critical for proper lymphatic vessel function. Congenital valve aplasia or agenesis can, in some cases, be a direct cause of lymphatic disease. Knowledge about the molecular mechanisms operating during the development and maintenance of LVs may thus aid in the establishment of novel therapeutic approaches to treat lymphatic disorders. In this study, we examined the role of Connexin43 (Cx43), a gap junction protein expressed in lymphatic endothelial cells (LECs), during valve development. Mouse embryos with a null mutation in Cx43 (Gja1) were previously shown to completely lack mesenteric LVs at embryonic day 18. However, interpreting the phenotype of Cx43-/- mice was complicated by the fact that global deletion of Cx43 causes perinatal death due to heart defects during embryogenesis. We have now generated a mouse model (Cx43?LEC) with a lymphatic-specific ablation of Cx43 and show that the absence of Cx43 in LECs causes a delay (rather than a complete block) in LV initiation, an increase in immature valves with incomplete leaflet elongation, a reduction in the total number of valves, and altered lymphatic capillary patterning. The physiological consequences of these lymphatic changes were leaky valves, insufficient lymph transport and reflux, and a high incidence of lethal chylothorax. These results demonstrate that the expression of Cx43 is specifically required in LECs for normal development of LVs.
Project description:Breast cancer metastasis involves lymphatic dissemination in addition to hematogenous spreading. Although stromal lymphatic vessels (LVs) serve as initial metastatic routes, roles of organ-residing LVs are underinvestigated. Here we show that lymphatic endothelial cells (LECs), a component of LVs within pre-metastatic niches, are conditioned by triple-negative breast cancer (TNBC) cells to accelerate metastasis. LECs within the lungs and lymph nodes, conditioned by tumour-secreted factors, express CCL5 that is not expressed either in normal LECs or in cancer cells, and direct tumour dissemination into these tissues. Moreover, tumour-conditioned LECs promote angiogenesis in these organs, allowing tumour extravasation and colonization. Mechanistically, tumour cell-secreted IL6 causes Stat3 phosphorylation in LECs. This pStat3 induces HIF-1? and VEGF, and a pStat3-pc-Jun-pATF-2 ternary complex induces CCL5 expression in LECs. This study demonstrates anti-metastatic activities of multiple repurposed drugs, blocking a self-reinforcing paracrine loop between breast cancer cells and LECs.
Project description:Wnt/?-catenin signaling is necessary for lymphatic vascular development. Oscillatory shear stress (OSS) enhances Wnt/?-catenin signaling in cultured lymphatic endothelial cells (LECs) to induce expression of the lymphedema-associated transcription factors GATA2 and FOXC2. However, the mechanisms by which OSS regulates Wnt/?-catenin signaling and GATA2 and FOXC2 expression are unknown. We show that OSS activates autocrine Wnt/?-catenin signaling in LECs in vitro. Tissue-specific deletion of Wntless, which is required for the secretion of Wnt ligands, reveals that LECs and vascular smooth muscle cells are complementary sources of Wnt ligands that regulate lymphatic vascular development in vivo. Further, the LEC master transcription factor PROX1 forms a complex with ?-catenin and the TCF/LEF transcription factor TCF7L1 to enhance Wnt/?-catenin signaling and promote FOXC2 and GATA2 expression in LECs. Thus, our work defines Wnt sources, reveals that PROX1 directs cell fate by acting as a Wnt signaling component, and dissects the mechanisms of PROX1 and Wnt synergy.
Project description:Emerging evidence suggests that intestinal stromal cells (IntSCs) play essential roles in maintaining intestinal homeostasis. However, the extent of heterogeneity within the villi stromal compartment and how IntSCs regulate the structure and function of specialized intestinal lymphatic capillary called lacteal remain elusive. Here we show that selective hyperactivation or depletion of YAP/TAZ in PDGFR?+ IntSCs leads to lacteal sprouting or regression with junctional disintegration and impaired dietary fat uptake. Indeed, mechanical or osmotic stress regulates IntSC secretion of VEGF-C mediated by YAP/TAZ. Single-cell RNA sequencing delineated novel subtypes of villi fibroblasts that upregulate Vegfc upon YAP/TAZ activation. These populations of fibroblasts were distributed in proximity to lacteal, suggesting that they constitute a peri-lacteal microenvironment. Our findings demonstrate the heterogeneity of IntSCs and reveal that distinct subsets of villi fibroblasts regulate lacteal integrity through YAP/TAZ-induced VEGF-C secretion, providing new insights into the dynamic regulatory mechanisms behind lymphangiogenesis and lymphatic remodeling.
Project description:Collecting lymphatic ducts contain intraluminal valves that prevent backflow. In mice, lymphatic valve morphogenesis begins at embryonic day 15.5 (E15.5). In the mesentery, Prox1 expression is high in valve-forming lymphatic endothelial cells, whereas cells of the lymphatic ducts express lower levels of Prox1. Integrin ?9, fibronectin EIIIA, Foxc2, calcineurin and the gap junction protein Cx37 are required for lymphatic valve formation. We show that Notch1 is expressed throughout the developing mesenteric lymphatic vessels at E16.5, and that, by E18.5, Notch1 expression becomes highly enriched in the lymphatic valve endothelial cells. Using a Notch reporter mouse, Notch activity was detected in lymphatic valves at E17.5 and E18.5. The role of Notch in lymphatic valve morphogenesis was studied using a conditional lymphatic endothelial cell driver either to delete Notch1 or to express a dominant-negative Mastermind-like (DNMAML) transgene. Deletion of Notch1 led to an expansion of Prox1(high) cells, a defect in Prox1(high) cell reorientation and a decrease in integrin ?9 expression at sites of valve formation. Expression of DNMAML, which blocks all Notch signaling, resulted in a more severe phenotype characterized by a decrease in valves, failure of Prox1(high) cells to cluster, and rounding of the nuclei and decreased fibronectin-EIIIA expression in the Prox1(high) cells found at valve sites. In human dermal lymphatic endothelial cells, activation of Notch1 or Notch4 induced integrin ?9, fibronectin EIIIA and Cx37 expression. We conclude that Notch signaling is required for proper lymphatic valve formation and regulates integrin ?9 and fibronectin EIIIA expression during valve morphogenesis.
Project description:Lymphatic endothelial cells (LECs) are differentiated from blood vascular endothelial cells (BECs) during embryogenesis and this physiological cell fate specification is controlled by PROX1, the master regulator for lymphatic development. When Kaposi sarcoma herpes virus (KSHV) infects host cells, it activates the otherwise silenced embryonic endothelial differentiation program and reprograms their cell fates. Interestingly, previous studies demonstrated that KSHV drives BECs to acquire a partial lymphatic phenotype by upregulating PROX1 (forward reprogramming), but stimulates LECs to regain some BEC-signature genes by downregulating PROX1 (reverse reprogramming). Despite the significance of this KSHV-induced bidirectional cell fate reprogramming in KS pathogenesis, its underlying molecular mechanism remains undefined. Here, we report that IL3 receptor alpha (IL3R?) and NOTCH play integral roles in the host cell type-specific regulation of PROX1 by KSHV. In BECs, KSHV upregulates IL3R? and phosphorylates STAT5, which binds and activates the PROX1 promoter. In LECs, however, PROX1 was rather downregulated by KSHV-induced NOTCH signal via HEY1, which binds and represses the PROX1 promoter. Moreover, PROX1 was found to be required to maintain HEY1 expression in LECs, establishing a reciprocal regulation between PROX1 and HEY1. Upon co-activation of IL3R? and NOTCH, PROX1 was upregulated in BECs, but downregulated in LECs. Together, our study provides the molecular mechanism underlying the cell type-specific endothelial fate reprogramming by KSHV.
Project description:Schlemm's canal (SC) is a specialized vascular structure in the eye that functions to drain aqueous humor from the intraocular chamber into systemic circulation. Dysfunction of SC has been proposed to underlie increased aqueous humor outflow (AHO) resistance, which leads to elevated ocular pressure, a factor for glaucoma development in humans. Here, using lymphatic and blood vasculature reporter mice, we determined that SC, which originates from blood vessels during the postnatal period, acquires lymphatic identity through upregulation of prospero homeobox protein 1 (PROX1), the master regulator of lymphatic development. SC expressed lymphatic valve markers FOXC2 and integrin ?9 and exhibited continuous vascular endothelial-cadherin (VE-cadherin) junctions and basement membrane, similar to collecting lymphatics. SC notably lacked luminal valves and expression of the lymphatic endothelial cell markers podoplanin and lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1). Using an ocular puncture model, we determined that reduced AHO altered the fate of SC both during development and under pathologic conditions; however, alteration of VEGF-C/VEGFR3 signaling did not modulate SC integrity and identity. Intriguingly, PROX1 expression levels linearly correlated with SC functionality. For example, PROX1 expression was reduced or undetectable under pathogenic conditions and in deteriorated SCs. Collectively, our data indicate that PROX1 is an accurate and reliable biosensor of SC integrity and identity.
Project description:Lymph is returned to the blood circulation exclusively via four lymphovenous valves (LVVs). Despite their vital importance, the architecture and development of LVVs is poorly understood. We analyzed the formation of LVVs at the molecular and ultrastructural levels during mouse embryogenesis and identified three critical steps. First, LVV-forming endothelial cells (LVV-ECs) differentiate from PROX1(+) progenitors and delaminate from the luminal side of the veins. Second, LVV-ECs aggregate, align perpendicular to the direction of lymph flow and establish lympho-venous connections. Finally, LVVs mature with the recruitment of mural cells. LVV morphogenesis is disrupted in four different mouse models of primary lymphedema and the severity of LVV defects correlate with that of lymphedema. In summary, we have provided the first and the most comprehensive analysis of LVV development. Furthermore, our work suggests that aberrant LVVs contribute to lymphedema.
2016-01-01 | S-EPMC4688075 | BioStudies
Project description:The lymphatic system is composed of a hierarchical network of fluid absorbing lymphatic capillaries and transporting collecting vessels. Despite distinct functions and morphologies, molecular mechanisms that regulate the identity of the different vessel types are poorly understood. Through transcriptional analysis of murine dermal lymphatic endothelial cells (LECs), we identified Foxp2, a member of the FOXP family of transcription factors implicated in speech development, as a collecting vessel signature gene. FOXP2 expression was induced after initiation of lymph flow in vivo and upon shear stress on primary LECs in vitro. Loss of FOXC2, the major flow-responsive transcriptional regulator of lymphatic valve formation, abolished FOXP2 induction in vitro and in vivo. Genetic deletion of Foxp2 in mice using the endothelial specific Tie2-Cre or the tamoxifen inducible LEC-specific Prox1-CreERT2 line resulted in enlarged collecting vessels and defective valves characterized by loss of NFATc1 activity. Our results identify FOXP2 as a new flow-induced transcriptional regulator of collecting lymphatic vessel morphogenesis and highlight the existence of unique transcription factor codes in the establishment of vessel-type specific endothelial cell identities.