Coagulation in Lymphatic System

The lymphatic system maintains homeostasis of the internal environment between the cells in tissues and the blood circulation. The coagulation state of lymph is determined by conditions of coagulation factors and lymphatic vessels. Internal obliteration, external compression or abnormally increased lymphatic pressure may predispose to localized lymphatic coagulation. In physiological conditions, an imbalance of antithrombin and thrombokinase reduces lymphatic thrombosis. However, the release of factor X by lymphatic endothelium injury may trigger coagulation casacade, causing blockage of lymphatic vessels and lymphedema. Heterogeneity of lymphatic vessels in various tissues may lead to distinct levels and patterns of coagulation in specific lymphatic vessels. The quantitative and qualitative measurement of clotting characteristic reveals longer time for clotting to occur in the lymph than in the blood. Cancer, infections, amyloidosis and lymph node dissection may trigger thrombosis in the lymphatic vessels. In contrast to venous or arterial thrombosis, lymphatic thrombosis has rarely been reported, and its actual prevalence is likely underestimated. In this review, we summarize the mechanisms of coagulation in lymphatic system, and discuss the lymphatic thrombosis-related diseases.

Lymphatic Clearance of Immune Cells in Cardiovascular Disease

Recent advances in our understanding of the lymphatic system, its function, development, and role in pathophysiology have changed our views on its importance. Historically thought to be solely involved in the transport of tissue fluid, lipids, and immune cells, the lymphatic system displays great heterogeneity and plasticity and is actively involved in immune cell regulation. Interference in any of these processes can be deleterious, both at the developmental and adult level. Preclinical studies into the cardiac lymphatic system have shown that invoking lymphangiogenesis and enhancing immune cell trafficking in ischaemic hearts can reduce myocardial oedema, reduce inflammation, and improve cardiac outcome. Understanding how immune cells and the lymphatic endothelium interact is also vital to understanding how the lymphatic vascular network can be manipulated to improve immune cell clearance. In this Review, we examine the different types of immune cells involved in fibrotic repair following myocardial infarction. We also discuss the development and function of the cardiac lymphatic vasculature and how some immune cells interact with the lymphatic endothelium in the heart. Finally, we establish how promoting lymphangiogenesis is now a prime therapeutic target for reducing immune cell persistence, inflammation, and oedema to restore heart function in ischaemic heart disease.

FOXC2 controls adult lymphatic endothelial specialization, function, and gut lymphatic barrier preventing multiorgan failure

The mechanisms maintaining adult lymphatic vascular specialization throughout life and their role in coordinating inter-organ communication to sustain homeostasis remain elusive. We report that inactivation of the mechanosensitive transcription factor Foxc2 in adult lymphatic endothelium leads to a stepwise intestine-to-lung systemic failure. Foxc2 loss compromised the gut epithelial barrier, promoted dysbiosis and bacterial translocation to peripheral lymph nodes, and increased circulating levels of purine metabolites and angiopoietin-2. Commensal microbiota depletion dampened systemic pro-inflammatory cytokine levels, corrected intestinal lymphatic dysfunction, and improved survival. Foxc2 loss skewed the specialization of lymphatic endothelial subsets, leading to populations with mixed, pro-fibrotic identities and to emergence of lymph node–like endothelial cells. Our study uncovers a cross-talk between lymphatic vascular function and commensal microbiota, provides single-cell atlas of lymphatic endothelial subtypes, and reveals organ-specific and systemic effects of dysfunctional lymphatics. These effects potentially contribute to the pathogenesis of diseases, such as inflammatory bowel disease, cancer, or lymphedema.

Shape and Function of Interstitial Chemokine CCL21 Gradients Are Independent of Heparan Sulfates Produced by Lymphatic Endothelium

Gradients of chemokines and growth factors guide migrating cells and morphogenetic processes. Migration of antigen-presenting dendritic cells from the interstitium into the lymphatic system is dependent on chemokine CCL21, which is secreted by endothelial cells of the lymphatic capillary, binds heparan sulfates and forms gradients decaying into the interstitium. Despite the importance of CCL21 gradients, and chemokine gradients in general, the mechanisms of gradient formation are unclear. Studies on fibroblast growth factors have shown that limited diffusion is crucial for gradient formation. Here, we used the mouse dermis as a model tissue to address the necessity of CCL21 anchoring to lymphatic capillary heparan sulfates in the formation of interstitial CCL21 gradients. Surprisingly, the absence of lymphatic endothelial heparan sulfates resulted only in a modest decrease of CCL21 levels at the lymphatic capillaries and did neither affect interstitial CCL21 gradient shape nor dendritic cell migration toward lymphatic capillaries. Thus, heparan sulfates at the level of the lymphatic endothelium are dispensable for the formation of a functional CCL21 gradient.

Unique functions for Notch4 in murine embryonic lymphangiogenesis

In mice, embryonic dermal lymphatic development is a well-understood system used to study the role of genes in physiological lymphangiogenesis. The Notch signaling is an evolutionary conserved pathway that modulates cell fate decisions and shown to both inhibit and promote dermal lymphangiogenesis. Here, we demonstrate distinct roles for Notch4 signaling versus canonical Notch signaling in embryonic dermal lymphangiogenesis. At E14.5, actively growing dermal lymphatics expressed NOTCH1, NOTCH4 and DLL4, with DLL4 expression strongest and Notch active in the lymphangiogenic sprouts. Treatment of cultured LECs with VEGF-A or VEGF-C upregulated Dll4 transcripts, but differentially regulated Notch1 and Notch4 expression, and the Notch effectors of the Hes/Hey families, suggesting that VEGF-A and VEGF-C distinctly modulate Dll4/Notch signaling in the lymphatic endothelium. Mice nullizygous for Notch4 had an increase in the closure of the lymphangiogenic fronts towards the midline which correlated with reduced vessel caliber in the maturing lymphatic plexus. Activation of Notch4 suppressed lymphatic endothelial cell migration in a wounding assay significantly more then Notch1 activation, suggesting a dominant role for Notch4 in LEC migration. Unlike Notch4 nulls, inhibition of canonical Notch signaling by ectopically expressing a dominant negative form of MAML1 (DNMAML) in Prox1+ lymphatic endothelium suppressed lymphatic endothelial cell proliferation consistent with what has been described for the loss of lymphatic endothelial Notch1. Moreover, loss of Notch4 did not disrupt lymphatic endothelial canonical Notch signaling. Thus, we propose that Notch4 signaling and canonical Notch signaling have distinct functions in the coordination of embryonic dermal lymphangiogenesis.

Tissue-resident macrophages regulate lymphatic vessel growth and patterning in the developing heart

ABSTRACTMacrophages are components of the innate immune system with key roles in tissue inflammation and repair. It is now evident that macrophages also support organogenesis, but few studies have characterized their identity, ontogeny and function during heart development. Here, we show that the distribution and prevalence of resident macrophages in the subepicardial compartment of the developing heart coincides with the emergence of new lymphatics, and that macrophages interact closely with the nascent lymphatic capillaries. Consequently, global macrophage deficiency led to extensive vessel disruption, with mutant hearts exhibiting shortened and mis-patterned lymphatics. The origin of cardiac macrophages was linked to the yolk sac and foetal liver. Moreover, the Cx3cr1+ myeloid lineage was found to play essential functions in the remodelling of the lymphatic endothelium. Mechanistically, macrophage hyaluronan was required for lymphatic sprouting by mediating direct macrophage-lymphatic endothelial cell interactions. Together, these findings reveal insight into the role of macrophages as indispensable mediators of lymphatic growth during the development of the mammalian cardiac vasculature.

Abstract 17346: Loss of Neuropilin 2 Impairs Lymphangiogenesis and Exacerbates Lymphedema

Dysfunctional lymphatic networks can lead to lymphatic diseases such as lymphedema as well as aggravate cardiovascular diseases such as atherosclerosis and hypertension. Some primary lymphedema patients have mutations in the Neuropilin 2 ( NRP2 ) gene, which encodes a transmembrane receptor that acts with VEGFR3 for the ligands VEGF-C and VEGF-D. Nrp2 has been shown to be a key regulator of lymphatic vessel development in the neonate as global Nrp2 -deficient mice suffer from reduced lymphatic density. However, Nrp2 protein expression is dramatically downregulated after birth and its role in adult lymphangiogenesis or its relevance in lymphedema is less clear. Our hypothesis is that Nrp2 expression is necessary for optimal lymphangiogenesis and proper lymphatic drainage function in the adult, and that loss of Nrp2 , either genetically or transcriptionally via down-regulation by specific transcription factors, may worsen lymphedema. In acute and chronic mouse inflammation models, we found that loss of Nrp2 specifically in the lymphatic endothelium ( prox1-cre ERT2 ;Nrp2 f/f ) resulted in prolonged swelling and reduced lymphatic drainage compared to control mice. Using the VEGFC-induced corneal lymphangiogenesis model and the tail wound-induced secondary lymphedema model, LEC-Nrp2-iKO mice showed diminished lymphangiogenesis and reduced lymphatic drainage compared to littermate controls. Furthermore, primary mouse dermal LEC isolated from Nrp2-iKO mice reveal suppressed VEGFR3 activation, signaling, and proliferation following VEGF-C stimulation, compared to LEC isolated from control mice. Collectively, our results identify Nrp2 as a critical mediator of lymphangiogenesis and homeostatic lymphatic drainage function in adult tissues.

Properties of rapamycin solid lipid nanoparticles for lymphatic access through the lungs & part II: the effect of nanoparticle charge

Aim: Lymphangioleiomyomatosis is characterized by smooth muscle-like cells in the lungs that spread to other organs via lymphatic vessels. Oral rapamycin is restricted by low bioavailability approximately 15%. The aim of the present study is to systematically investigate the effect of inhaled rapamycin solid lipid nanoparticles (Rapa-SLN) surface charge on efficacy and penetration into the lymphatics. Materials & methods: Rapa-SLN formulations with different charge: neutral, positive and negative, were produced and assessed for their physicochemical particle characteristics and efficacy in vitro. Results: Negative Rapa-SLNs were significantly faster at entering the lymphatic endothelium and more potent at inhibiting lymphanigiogenesis compared with neutral and positive Rapa-SLNs. Conclusion: Negative Rapa-SLNs showed efficient lymphatic access and should therefore be investigated further as a treatment for targeting extrapulmonary lymphangioleiomyomatosis.