DLL4 promotes continuous adult intestinal lacteal regeneration and dietary fat transport.

The small intestine is a dynamic and complex organ that is characterized by constant epithelium turnover and crosstalk among various cell types and the microbiota. Lymphatic capillaries of the small intestine, called lacteals, play key roles in dietary fat absorption and the gut immune response; however, little is known about the molecular regulation of lacteal function. Here, we performed a high-resolution analysis of the small intestinal stroma and determined that lacteals reside in a permanent regenerative, proliferative state that is distinct from embryonic lymphangiogenesis or quiescent lymphatic vessels observed in other tissues. We further demonstrated that this continuous regeneration process is mediated by Notch signaling and that the expression of the Notch ligand delta-like 4 (DLL4) in lacteals requires activation of VEGFR3 and VEGFR2. Moreover, genetic inactivation of Dll4 in lymphatic endothelial cells led to lacteal regression and impaired dietary fat uptake. We propose that such a slow lymphatic regeneration mode is necessary to match a unique need of intestinal lymphatic vessels for both continuous maintenance, due to the constant exposure to dietary fat and mechanical strain, and efficient uptake of fat and immune cells. Our work reveals how lymphatic vessel responses are shaped by tissue specialization and uncover a role for continuous DLL4 signaling in the function of adult lymphatic vasculature

DLL4 promotes continuous adult intestinal lacteal regeneration and dietary fat transport

The small intestine is a dynamic and complex organ that is characterized by constant epithelium turnover and crosstalk among various cell types and the microbiota. Lymphatic capillaries of the small intestine, called lacteals, play key roles in dietary fat absorption and the gut immune response; however, little is known about the molecular regulation of lacteal function. Here, we performed a high-resolution analysis of the small intestinal stroma and determined that lacteals reside in a permanent regenerative, proliferative state that is distinct from embryonic lymphangiogenesis or quiescent lymphatic vessels observed in other tissues. We further demonstrated that this continuous regeneration process is mediated by Notch signaling and that the expression of the Notch ligand delta-like 4 (DLL4) in lacteals requires activation of VEGFR3 and VEGFR2. Moreover, genetic inactivation of Dll4 in lymphatic endothelial cells led to lacteal regression and impaired dietary fat uptake. We propose that such a slow lymphatic regeneration mode is necessary to match a unique need of intestinal lymphatic vessels for both continuous maintenance, due to the constant exposure to dietary fat and mechanical strain, and efficient uptake of fat and immune cells. Our work reveals how lymphatic vessel responses are shaped by tissue specialization and uncover a role for continuous DLL4 signaling in the function of adult lymphatic vasculature

Mechanosensitive ACKR4 scavenges CCR7 chemokines to facilitate T cell de-adhesion and passive transport by flow in inflamed afferent lymphatics.

T cell migration via afferent lymphatics to draining lymph nodes (dLNs) depends on expression of CCR7 in T cells and CCL21 in the lymphatic vasculature. Once T cells have entered lymphatic capillaries, they slowly migrate into contracting collecting vessels. Here, lymph flow picks up, inducing T cell detachment and rapid transport to the dLNs. We find that the atypical chemokine receptor 4 (ACKR4), which binds and internalizes CCL19 and CCL21, is induced by lymph flow in endothelial cells lining lymphatic collectors, enabling them to scavenge these chemokines. In the absence of ACKR4, migration of T cells to dLNs in TPA-induced inflammation is significantly reduced. While entry into capillaries is not impaired, T cells accumulate in the ACKR4-deficient dermal collecting vessel segments. Overall, our findings identify an ACKR4-mediated mechanism by which lymphatic collectors facilitate the detachment of lymph-borne T cells in inflammation and their transition from crawling to free-flow toward the dLNs

FOXC2 and fluid shear stress stabilize postnatal lymphatic vasculature.

Biomechanical forces, such as fluid shear stress, govern multiple aspects of endothelial cell biology. In blood vessels, disturbed flow is associated with vascular diseases, such as atherosclerosis, and promotes endothelial cell proliferation and apoptosis. Here, we identified an important role for disturbed flow in lymphatic vessels, in which it cooperates with the transcription factor FOXC2 to ensure lifelong stability of the lymphatic vasculature. In cultured lymphatic endothelial cells, FOXC2 inactivation conferred abnormal shear stress sensing, promoting junction disassembly and entry into the cell cycle. Loss of FOXC2-dependent quiescence was mediated by the Hippo pathway transcriptional coactivator TAZ and, ultimately, led to cell death. In murine models, inducible deletion of Foxc2 within the lymphatic vasculature led to cell-cell junction defects, regression of valves, and focal vascular lumen collapse, which triggered generalized lymphatic vascular dysfunction and lethality. Together, our work describes a fundamental mechanism by which FOXC2 and oscillatory shear stress maintain lymphatic endothelial cell quiescence through intercellular junction and cytoskeleton stabilization and provides an essential link between biomechanical forces and endothelial cell identity that is necessary for postnatal vessel homeostasis. As FOXC2 is mutated in lymphedema-distichiasis syndrome, our data also underscore the role of impaired mechanotransduction in the pathology of this hereditary human disease

GATA2 is required for lymphatic vessel valve development and maintenance.

Heterozygous germline mutations in the zinc finger transcription factor GATA2 have recently been shown to underlie a range of clinical phenotypes, including Emberger syndrome, a disorder characterized by lymphedema and predisposition to myelodysplastic syndrome/acute myeloid leukemia (MDS/AML). Despite well-defined roles in hematopoiesis, the functions of GATA2 in the lymphatic vasculature and the mechanisms by which GATA2 mutations result in lymphedema have not been characterized. Here, we have provided a molecular explanation for lymphedema predisposition in a subset of patients with germline GATA2 mutations. Specifically, we demonstrated that Emberger-associated GATA2 missense mutations result in complete loss of GATA2 function, with respect to the capacity to regulate the transcription of genes that are important for lymphatic vessel valve development. We identified a putative enhancer element upstream of the key lymphatic transcriptional regulator PROX1 that is bound by GATA2, and the transcription factors FOXC2 and NFATC1. Emberger GATA2 missense mutants had a profoundly reduced capacity to bind this element. Conditional Gata2 deletion in mice revealed that GATA2 is required for both development and maintenance of lymphovenous and lymphatic vessel valves. Together, our data unveil essential roles for GATA2 in the lymphatic vasculature and explain why a select catalogue of human GATA2 mutations results in lymphedema

Molecular mechanisms of lymphatic vascular maintenance and repair

Lymphatic vessels maintain fluid homeostasis, transport dietary lipids and vitamins and contribute to immune surveillance by transporting soluble antigens and immune cells. Lymphatic vessels are also involved in many pathological conditions such as tumor metastasis and various inflammatory conditions. Dysfunction of lymphatic vessels leads to lymphedema, an accumulation of protein-rich fluid in interstitial space, resulting in swelling and fibrosis of the affected area. During the last decade signaling mechanisms involved in lymphatic vessel growth during development or tissue régénération have been a subject of intense research. Vascular Endothelial Growth Factor C (VEGFC) was identified as a key ligand that binds to lymphatic endothelial cell (LEC)-specific receptor tyrosine kinase, vascular endothelial growth factor receptor (VEGFR3), and induces LEC survival, prolifération and migration. In contrast, little is known about the pathways involved in lymphatic vessel maintenance and repair in adulthood. Adult lymphatic vasculature is considered to be essentially quiescent, with the exception of intestinal lymphatic capillaries. Intriguingly, accumulating clinical reports link the use of rapamycin to the development of leg or arm lymphedema. Rapamycin, which is an inhibitor of mechanistic target of rapamycin (mTOR), is commonly used in kidney transplant patients to prevent graft rejection. Lymphedema was not observed in patients treated with other immunosuppressive regimens, such as calcineurin inhibitors. In this study, we have investigated the mechanisms in lymphatic vascular maintenance and repair. Our data show that main place for LEC renewal in mature adult vasculature is lymphatic collecting vessels and proliferating cells are mostly concentrated in valve région. Our work on mTOR demonstrates a new activation stimulus for mTOR signaling, which is flow shear stress. mTOR is found to be indispensable for LECs under shear stress and mTOR activity is required for protein synthesis and extracellular matrix (ECM) production, thus ensuring the integrity of LEC monolayer. This phenotype we have observed in LECs might explain the lymphedema observed in rapamycin treated transplant patients. Further studies will be performed to illuminate upstream and downstream molecular partners of mTOR signaling in lymphatics

Palmdelphin Regulates Nuclear Resilience to Mechanical Stress in the Endothelium

Background: Palmdelphin (PALMD) belongs to the family of Paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms (SNPs) in the PALMD locus that result in reduced expression are strong risk factors for development of calcific aortic valve stenosis (CAVS) and predict severity of the disease. Methods: Immunodetection and public database screening showed dominant expression of PALMD in endothelial cells (ECs) in brain and cardiovascular tissues including aortic valves. Mass spectrometry, co-immunoprecipitation and immunofluorescent staining allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in siRNA-treated EC cultures, in knockout mice, and human valve samples. RNA sequencing of ECs and transcript arrays on valve samples from an aortic valve study cohort including patients with the SNP rs7543130, informed about gene regulatory changes. Results: ECs express the cytosolic PALMD-KKVI splice variant, which associated with RAN GTPase activating protein1 (RANGAP1). RANGAP1 regulates the activity of the GTPase RAN and thereby, nucleocytoplasmic shuttling via Exportin1 (XPO1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, and nuclear arrest of the XPO1 cargoes p53 and p21. This indicates an important role for PALMD in nucleocytoplasmic transport and consequently, in gene regulation due to the impact on localization of transcriptional regulators. Changes in EC responsiveness upon loss of PALMD expression included failure to form a perinuclear actin cap when exposed to flow, indicating lack of protection against mechanical stress. Loss of the actin cap correlated with misalignment of the nuclear long axis relative to the cell body, observed in PALMD-deficient ECs, Palmd−/− mouse aorta and human aortic valve samples derived from CAVS patients. In agreement with these changes in EC behavior, gene ontology analysis showed enrichment of nuclear- and cytoskeleton-related terms in PALMD-silenced ECs. Conclusions: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex alters the subcellular localization of RANGAP1 and XPO1, and leads to nuclear arrest of the XPO1 cargoes p53 and p21, accompanied by gene regulatory changes and loss of actin-dependent nuclear resilience. Combined, these consequences of reduced PALMD expression provide a mechanistic underpinning for PALMD's contribution to CAVS pathology

Palmdelphin Regulates Nuclear Resilience to Mechanical Stress in the Endothelium

Background: PALMD (palmdelphin) belongs to the family of paralemmin proteins implicated in cytoskeletal regulation. Single nucleotide polymorphisms in the PALMD locus that result in reduced expression are strong risk factors for development of calcific aortic valve stenosis and predict severity of the disease. Methods: Immunodetection and public database screening showed dominant expression of PALMD in endothelial cells (ECs) in brain and cardiovascular tissues including aortic valves. Mass spectrometry, coimmunoprecipitation, and immunofluorescent staining allowed identification of PALMD partners. The consequence of loss of PALMD expression was assessed in small interferring RNA-treated EC cultures, knockout mice, and human valve samples. RNA sequencing of ECs and transcript arrays on valve samples from an aortic valve study cohort including patients with the single nucleotide polymorphism rs7543130 informed about gene regulatory changes. Results: ECs express the cytosolic PALMD-KKVI splice variant, which associated with RANGAP1 (RAN GTP hydrolyase activating protein 1). RANGAP1 regulates the activity of the GTPase RAN and thereby nucleocytoplasmic shuttling via XPO1 (Exportin1). Reduced PALMD expression resulted in subcellular relocalization of RANGAP1 and XPO1, and nuclear arrest of the XPO1 cargoes p53 and p21. This indicates an important role for PALMD in nucleocytoplasmic transport and consequently in gene regulation because of the effect on localization of transcriptional regulators. Changes in EC responsiveness on loss of PALMD expression included failure to form a perinuclear actin cap when exposed to flow, indicating lack of protection against mechanical stress. Loss of the actin cap correlated with misalignment of the nuclear long axis relative to the cell body, observed in PALMD-deficient ECs, Palmd(-/-) mouse aorta, and human aortic valve samples derived from patients with calcific aortic valve stenosis. In agreement with these changes in EC behavior, gene ontology analysis showed enrichment of nuclear- and cytoskeleton-related terms in PALMD-silenced ECs. Conclusions: We identify RANGAP1 as a PALMD partner in ECs. Disrupting the PALMD/RANGAP1 complex alters the subcellular localization of RANGAP1 and XPO1, and leads to nuclear arrest of the XPO1 cargoes p53 and p21, accompanied by gene regulatory changes and loss of actin-dependent nuclear resilience. Combined, these consequences of reduced PALMD expression provide a mechanistic underpinning for PALMD's contribution to calcific aortic valve stenosis pathology