The Lymphatic Vasculature in the 21st Century: Novel Functional Roles in Homeostasis and Disease

Abstract

Mammals have two specialized vascular circulatory systems: the blood vasculature and the lymphatic vasculature. The lymphatic vasculature is a unidirectional conduit that returns filtered interstitial arterial fluid and tissue metabolites to the blood circulation. It also plays major roles in immune cell trafficking and lipid absorption. As we discuss in this review, the molecular characterization of lymphatic vascular development and our understanding of this vasculature's role in pathophysiological conditions has greatly improved in recent years, changing conventional views about the roles of the lymphatic vasculature in health and disease. Morphological or functional defects in the lymphatic vasculature have now been uncovered in several pathological conditions. We propose that subtle asymptomatic alterations in lymphatic vascular function could underlie the variability seen in the body's response to a wide range of human diseases.

Keywords: Alzheimer's; Crohn's disease; Lymphatics; Parkinson's; cardiovascular; glaucoma; immunity; inflammation; lymphatic function; lymphatic vasculature; lymphedema; myocardial infarction; neurological disease; obesity; tumors.

Figure 1:. Overview of the main structures forming the mammalian lymphatic system:

A) The lymphatic vasculature (green) is an arborized network that runs in parallel to the blood vasculature (red and blue). Smaller caliber initial lymphatics absorb the fluid that continuously leaks out from the blood capillary bed and drain their contents (lymph) into larger caliber collecting lymphatics specialized for transport. Lymph is filtered through lymph nodes before entering thoracic or right lymphatic ducts, which returns the lymph to the blood circulation via two pairs of bilaterally located lymphovenous valves. B) Initial lymphatics comprise a single layer of loosely connected lymphatic endothelial cells (LECs) lacking a continuous basement membrane and perivascular mural cells. LECs within initial lymphatics are inter-connected through discontinuous button-like junctions that facilitate the uptake of interstitial fluid and macromolecules released by the blood vasculature. C) In collecting lymphatics, LECs are connected to each other through tighter, continuous zipper-like junctions, and are covered with specialized muscle cells (SMC) that provide contractile activity to assist lymph flow. Collecting lymphatic vessels have valves that regulate the unidirectional flow of lymph, as well as a coordinated contraction of muscle cells that facilitates the transport of lymph back to the blood circulation. D) The lymph nodes (more than 200 in humans), along stretches of collecting lymphatics, are highly organized with segregated compartments of B and T lymphocytes, with lymphatic endothelial cells helping to form the subcapsular and medullary sinuses. The nodes are fed by blood vessels with a specialized postcapillary venule called the high endothelial venule that allows cellular entry into nodes via the blood, in addition to afferent lymphatic cell entry. E) the absorption of dietary fats and fat-soluble vitamins is dependent on intestinal villi, finger-like, enterocyte-lined extensions of the gut wall, containing a blood capillary network (red/blue) and one or two central lymphatic vessels (green) termed lacteals. In mammals, dietary lipids are repackaged in enterocytes into large triglyceride-loaded particles or “chylomicrons” which are taken up by the lacteals.

Figure 2:. Key events underlying construction of the mammalian lymphatic vasculature during embryonic development.

LEC specification and formation of LEC progenitors is initiated upon SOX18 and COUPTFII-mediated induction of Prox1 expression in embryonic venous endothelial cells, initially in the cardinal veins (CV). In turn, Prox1 activates the expression of Vegfr3, and Vegfr3 regulates Prox1 by establishing a feedback loop necessary to maintain the identity of LEC progenitors. Vegfc-mediated activation of Vegfr3 signaling is necessary to maintain Prox1 expression in LEC progenitors. Proteolytic processing of Vegfc by Ccbe1 and Adamts3 is required for lymphatic development to occur. Prox1+ LEC progenitors subsequently bud off from the CV and intersomitic vessels (ISV) and start to express differentiation markers such as podoplanin (Pdpn). Additional LEC progenitor cell sources contributing to lymphatic vasculature formation in other tissues include hemogenic endothelium in the case of mesenteric lymphatics, the second heart field in the case of ventral cardiac lymphatics and a group of cells within the dermal blood capillary bed which contribute to the dorsal dermal lymphatic vasculature. The initial lymphatic plexus is progressively arborized and lumenized and once flow within the network is initiated, valve development, crucial for unidirectional flow begins. Changes in cell polarity and the deposition of extracellular matrix are crucial for the generation of functional valves (shown in organge). An integral stage of lymphatic vessel maturation involves the recruitment of lymphatic muscle cells (magenta to the collecting lymphatics, acting to propel lymph back to the bloodstream. Schematic representing the lymphovenous valves. Most Prox1-expressing LEC progenitors bud off from the veins; however, a small subpopulation remains and forms the lymphovenous valves at the junction of the jugular and subclavian veins (SCV). Each of the valve’s two leaflets has two layers of PROX1+ ECs: an inner PROX1+/ PDPN+ layer continuous with the lymph sac and an outer PROX1+/PDPN− layer continuous with the veins. Left: The region of an E13.5 embryo in which the jugular and subclavian veins join to form the lymphovenous valves. Right: A frontal view of the boxed region shown at left. EJV, external jugular vein; IJV, internal jugular vein; LS, lymph sac; LV, lymphovenous valve; SVC, superior vena cava

Figure 3.. Traditional lymphatic-associated processes.

Lymphatic malfunction leads to vascular malformations, and primary and secondary lymphedema. Lymphedema is a disfiguring, disabling, and occasionally life-threatening disease that is characterized by fluid accumulation and the chronic and disabling swelling of the extremities. Dilated damaged and leaky lymphatics do not support the normal flow of lymph and promote unilateral edema and increased adipose tissue accumulation in the affected leg. Lymphatic vessels serve as a route by which tumor cells metastasize. Tumor lymphangiogenesis induced at the location of the primary tumor can facilitate the entry of metastatic tumor cells into lymphatic vessels and lymph nodes, while also supporting immunity to tumors for immune-mediated rejection. Lymphatic function is also important during immune and inflammatory responses. Initial lymphatics composed of a single layer of loosely connected LECs (in green) lack a continuous basement membrane and lymphatic muscle cells. These vessels are highly permeable to interstitial fluid and macromolecules, pathogens, and immune cells (i.e, leukocytes, neutrophils, macrophages). The initial lymphatics drain into larger collecting lymphatics in which LECs are connected to each other through tighter, continuous zipper-like junctions, are covered with muscle-like cells and have valves that regulate the unidirectional flow of lymph.

Figure 4.. Schematic representation of the major novel functional roles of the lymphatic vasculature in health and disease.

Recent work has uncovered important roles for the lymphatic vasculature in normal and pathological processes. Conditions in which lymphatic vessels are implicated include obesity, IBD/Crohn’s disease, cardiovascular pathologies (atherosclerosis and myocardial infarction), glaucoma and neurological diseases (Alzheimer’s, Parkinson’s, stroke and brain trauma, multiple sclerosis and brain tumors, age-related cognitive decline). In obesity, a direct link between asymptomatic defective and leaky lymphatics and increased adipogenesis and obesity was demonstrated so far in mice (Prox1^+/−). It is possible that similar alterations are also responsible in certain forms of obesity in humans. In Crohn’s disease, lymphatics proliferate at the inflamed gut wall where creeping fat is frequently also observed, but this proliferation is counterbalanced by leukocyte-rich obstructions present in the collecting lymphatics. These regions may also be leaky, and leakiness as discussed for obesity, in turn, may drive creeping fat. Crohn’s disease patients with the least lymphatic expansion are more likely to experience relapse after bowel resection. Recent studies suggested a beneficial role for lymphatics in restoring heart function after cardiac injury and that cardiac lymphatic vessels could be therapeutic targets to restore cardiac function after injury. In cardiovascular diseases, atherosclerosis, characterized by the accumulation of plaques comprising fat, cholesterol and immune cells inside the arterial vessel wall, results in the narrowing and hardening of arterial walls, limiting blood flow from the heart. Lymphatics are found at atherosclerotic sites in the adventitial layer of coronary arteries. Myocardial infarction is a life-threatening condition that occurs when blood flow to the heart abruptly cuts off, usually as a consequence of blockage in the coronary arteries, resulting in tissue damage and massive cardiomyocyte death that leads to the formation of fibrotic tissue, pathological remodeling and eventually heart failure. In glaucoma, resistance to aqueous humor outflow is increased, reducing drainage through the Schlemm’s canal and resulting in elevated intraocular pressure and optic neuropathy. Lymphatics are also important novel players in a variety of neurological disorders. It is likely that additional novel functional roles of the lymphatic vasculature in normal and pathological settings will be identified.

Figure 5:. A role for lymph nodes in the containment of tumor metastases and infection.

A traditional function of lymphatic vessels is their association with lymph nodes. More detailed structure of lymph nodes is shown in Figures 1 and 3. As shown in the left panel, lymph nodes effectively contain pathogens, in part by coordinating the recruitment of neutrophils through high endothelial venules of the blood stream that, separately from lymphatics, invest lymph nodes. Lymph nodes also serve a key role in containing activated antigen-presenting cells that express tissue factor (factor 3, F3) that functions to initiate coagulation. Finally, lymph nodes often limit metastases that arrive to the lymph node; that is, many metastases to lymph nodes are stopped therein and do not account for distal spread of the tumor. Each putative, distinct tumor clone is shown in a separate color. Right panel depicts outcomes that can occur if lymph node containment dramatically fails. The resulting severe disease risks that emerge under these conditions include enhanced tumor spread, sepsis and spread of infection from organ to organ, and infection-associated disseminated intravascular coagulation (DIC) that results from failed lymph node containment of F3-expressing antigen-presenting cells and their arrival to blood, as seen in Ebola virus infection.

Figure 6.. Lymphatic/glymphatic connection in health and disease.

(A) The brain is a highly active organ and its waste products (metabolites, cellular debris, misfolded proteins) need to be removed. The most recently proposed mechanism for brain waste disposal is the glymphatic pathway, which refers to a framework for fluid flow through the brain parenchyma. Cerebrospinal fluid (CSF; depicted in light blue around the brain) is produced by the epithelial cells of the choroid plexus within the brain’s ventricles and circulates within the subarachnoid space. At the brain surface the meningeal vasculature dives into the brain and through these para-arterial spaces the CSF follows a path towards the parenchyma. Pulsating arteries propel CSF through the astroglial endfeet into the parenchyma. This process drives the efflux of brain interstitial fluid, carrying metabolites, protein aggregates, and other waste products from the parenchyma along para-venous walls, back into the CSF. Finally, CSF ends up in the dura mater and this cellular and molecular waste is drained by meningeal lymphatic vessels into the deep cervical lymph nodes. Normal meningeal lymphatic drainage ensures proper clearance of brain waste, through maintaining appropriate glymphatic function. However, disrupted meningeal lymphatics may underlie several neurological disorders. (B) Aging is characterized by dysfunction of many vital systems, including the lymphatic vasculature. A typical characteristic of the age-related deterioration observed in meningeal lymphatics is their reduced diameter and branching, accompanied by impaired drainage into the deep cervical lymph nodes (dCLNs). Impaired meningeal lymphatic function may underlie accumulation and aggregation of proteins, exacerbating conditions such as Alzheimer’s disease, Parkinson’s disease, and others. Poorly functional meningeal lymphatics may also impede immune response against brain tumors. Overactive, meningeal lymphatics, however, may result in break of CNS immune privilege, leading to pathological neuroinflammation, associated with multiple sclerosis