Abstract

INTRODUCTION

Rising interest in the lymphatic system over the past thirty years, combined with advances in imaging, cell, and molecular biology, and genetics have enabled a significant expanse in knowledge about lymphatic vessels and the cellular and molecular mechanisms underlying lymphedema formation. Substantial advances concerning the unique sets of genes and molecules that drive lymphatic vessel development and maturation have been achieved, as well as notable new knowledge pertaining to the physiology of lymph propulsion. Still, the persistence of lymphedema as a disease with limited treatment options motivates the need to reconsider what we know and do not know about how fluid enters lymphatic vessels. To do this, an integrated view along the path of fluid flow from blood capillaries to the sites of fluid uptake into initial lymphatic vessels (also known as lymphatic capillaries) is needed.

Since the discovery of VEGF-C/VEGFR-3 signaling and its connection to lymphatic endothelial cells [1, 2], lymphatic biology has experienced a resurgence with research focusing on where, when, and how lymphatic vessels form and function during the developmental and post-developmental stages of life. Concepts that had not been broadly accepted previously, such as the importance of lymphatic function for central nervous system homeostasis, have been highlighted by modern techniques demonstrating lymphatic vessels in the meninges of the brain [3, 4]. Moreover, links between lymphatic structure and function to cancer metastases, neurodegenerative diseases, and cardiometabolic risk factors emphasize the importance of the lymphatic system well past tissue edema [5–11]. Recognizing that current understanding is still in an infancy, opportunities exist to establish new paradigms. A crucial perspective is gained from studying the blood microvascular-lymphatic interface. The objective of this review is to highlight the coordinated functional relationships between blood and lymphatic vessels in adult microvascular networks. Undoubtedly, understanding how fluid flows into and through a lymphatic network depends on coordination with the blood microvasculature. Through the consideration of tissue fluid homeostasis and the roles of microvascular leakage of plasma, initial lymphatic endothelial cell structure, and lymphatic/blood vessel patterning, this review identifies knowledge gaps that can guide future work at the functional interface between the microvascular blood and lymphatic network.

THE CIRCULATORY AND LYMPHATIC SYSTEMS AND TISSUE HOMEOSTASIS

Water balance is the most important regulatory mechanism of homeostatic function. In humans, body fluids contribute to about 70% of body weight, divided among the intracellular fluids within cells occupying about two-thirds of total fluid, and extracellular fluid, made up of blood plasma, interstitial fluid, and lymph. At the whole-body level, autonomic control of cardiovascular, renal, and gastrointestinal mechanisms governs water balance. At the microscopic level, microvascular barrier function regulates flow of plasma into tissues to form interstitial fluid. For cells in the tissues, regulated water transport across the plasma membrane is permitted by the thirteen isoforms of aquaporins (AQPs) that serve as pores for water to enter and exit cells, but not ions or larger charged solutes [12, 13]. In addition, ion channel compositions in combination with local ATP-driven transporters produce local ion gradients, with intracellular fluid generally rich in K⁺ and extracellular fluid rich in Na⁺ and Cl⁻ [14]. In mammals, this delicate set of homeostatic mechanisms requires a steady input of fluid containing nutrients from the microcirculation, and a reliable lymphatic drainage system that can remove excess fluid and the waste products that do not easily diffuse back into blood vessels.

The circulatory system carries oxygen and nutrients to tissues and removes the bulk of metabolic waste, in a continuous circuit consisting of arteries, arterioles, capillaries, venules, and veins. While a closed loop, this system is also inherently leaky in the microvascular beds, specifically at the capillary and postcapillary venules, where oxygen, water, and nutrients are delivered to the surrounding tissues [15]. This leakiness is best described as a consequence of the microvasculature being permeable to plasma proteins, thus resulting in a lack of Starling equilibria and a constant steady state level of filtration in the tissue microenvironment [16]. This net filtration into the tissue produces an increase in interstitial volume resulting in a need for drainage of the tissues to prevent swelling, which is normally fulfilled by the lymphatic system [17]. Unlike the circulatory system, the lymphatic system has a unidirectional organization like the root system of a plant, with blind-ended vessels that coalesce into larger vessels, leading to lymph nodes and larger lymph trunks that return extravasated fluids to the circulatory system [18].

Endothelial cells form the inner linings of all blood and lymphatic vessels. Capillaries represent the most abundant vessel type and are the primary exchange site of content between the vascular lumen and tissue. Blood and lymphatic capillaries are composed of a single layer of endothelial cells, forming a semi-permeable barrier between the capillary lumen and interstitial spaces. Lymphatic and blood endothelial cells are the primary players in vascular growth and regulate the exchange of gases, fluids, nutrients, signaling molecules and other cells in virtually all tissues in the body [19]. However, the specific mechanisms guiding or regulating fluid entry into lymphatic capillaries remain poorly understood [20]. The presence of one-way primary valves at endothelial cell junctions suggests that local pressures direct uptake, yet the observation of inter-endothelial cell gaps raise questions about pressure differences across the lymphatic vessel wall and highlight the potential dynamic nature of endothelial cell junctions to regulate fluid exchange.

Understanding the process of lymph formation indeed requires consideration of the structures and functions of both the circulatory and lymphatic systems. In the circulatory system, the network consists of the aorta and large arteries, which serve as conduits, followed by smaller arteries and arterioles that serve as resistance vessels that control blood flow to downstream capillary beds. The capillaries act as the exchange microvessels for oxygen diffusion and delivery of water and small solutes, while postcapillary venules act as the main site of plasma protein leakage and leukocyte extravasation into tissues [15]. The larger collecting venules and veins then serve as a capacitance system, essentially a reservoir of blood available for the heart to pump through the pulmonary circulation and then back into the aorta. Like the circulatory system, lymphatics have a hierarchy of vessels that serve different functions (Fig. 1). The point of interstitial fluid entry into the lymphatic system is the blind-ended lymphatic capillaries, also known as initial lymphatics. The balance of hydrostatic and osmotic pressures known as Starling forces help direct fluid from the capillaries into the interstitial space and toward the lymphatics [16, 17]. The entry of interstitial fluid into the initial lymphatic network, known as lymph formation, is thought to involve a unique discontinuous junction structure on the lymphatic capillary endothelium commonly referred to as “buttons” [21, 22]. Additional factors, such as the irregular shape of lymphatic capillaries may also contribute, according to results from computational models [23]. Lymphatic capillaries also serve as the main site for entry of antigen-presenting cells and lymphocytes [24–26]. The lymph, including fluid and cells, flows into conduit lymphatic vessels. While the mechanisms driving downstream flow through initial lymphatic vessels remains to be debated, mechanical forces have been associated with periodic suction forces produced downstream in the network [27]. Alternative suggested sources of mechanical forces include extracellular matrix deformation due to nearby arteriole vasomotion [20] and in case of the bat wing initial lymphatics, smooth muscle coverage of these special initial lymphatics give them the ability to contract [28, 29]. In the conduit lymphatic vessels, the button junctions are replaced by continuous junctions (“zippers”), and these vessels also have bicuspid intraluminal valves to prevent backflow but allow forward flow when pressure gradients favor propulsion of lymph forward through the system. Lymphatic vessels that contain intraluminal valves but either lack a smooth muscle layer or display intermittent coverage are called pre-collectors. It is currently unclear whether pre-collectors might serve as additional sites of lymph formation. When a more consistent smooth muscle layer is present, the vessels are referred to as collecting lymphatics. The smooth muscle has intrinsic contractile properties that serve to pump lymph. Combined with the intraluminal valves that prevent backflow, the contractions of the collecting lymphatics are likely the source of the periodic suction force that pulls lymph forward through the lymphatic capillaries and pre-collectors [20, 30]. The collecting lymphatics connect to lymph nodes and coalesce into larger lymph trunks. A significant portion of both water and innate immune cells found in prenodal lymph is returned to the circulatory system at lymph nodes where there is significant AQP-1 expression on both lymphatic and blood endothelial cells [31–36]. The remaining post-nodal lymph is returned to the circulation by the larger trunks, namely the thoracic duct and the right lymph duct.

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Fig 1.

Lymph Flow Through Lymphatic Networks Starts at the Interface with Blood Vessels in Microvascular Networks.

Lymph is formed when interstitial fluid enters lymphatic capillaries, also termed initial lymphatics (left image, thin arrows). The initial lymphatic networks drain into collecting lymphatic vessels, which are wrapped by smooth muscle cells. Phasic contraction of the smooth muscle layer is critical for lymphatic pumping (thick arrows). The intraluminal, secondary valve leaflets prevent segmental backflow. Consideration of fluid entrance into initial lymphatic vessels in microvascular networks requires an appreciation of the structural and functional interrelationships between lymphatic and blood vessel networks (right image). Plasma that leaks from the capillaries and postcapillary venules forms interstitial fluid, which flows through tissues and enters initial lymphatics. Immune cells also extravasate at postcapillary venules and enter the initial lymphatic network. The specific structures mediating entry of fluid and leukocytes remain an intensive area of investigation. Printed with permission from Anita Impagliazzo.

Imbalances in blood flow and the normal leakage of plasma can disrupt optimal interstitial fluid flow within tissues. Too little plasma leakage leads to dehydration, while excessive plasma leakage can cause edema. Under normal conditions, the microvascular leakage of fluids and solutes, also called capillary filtration, is considered equal to lymph flow. However, there are several scenarios in which the permeability of the postcapillary venule endothelium becomes high, and extravasation of fluids and plasma proteins temporarily exceeds the drainage capacity of lymphatic vessels, causing swelling (edema). This commonly occurs locally with insect bites and musculoskeletal injuries, which cause a local inflammatory response that typically resolves over time. However, shock caused by traumatic injury causes significant systemic microvascular leakage [37–40], and diseases such as diabetes increase microvascular permeability over the long-term, leading to chronic swelling [41]. Long-term venous diseases can increase venous hydrostatic pressure, which in turn increases capillary hydrostatic pressure, causing long-term edema. In some cases, the lymphatic system has normal clearance function but is simply overwhelmed, but eventually tissue fluid balance returns to normal. However, when lymphatic insufficiency is present, even a gradual increase in interstitial fluid can manifest into lymphedema, at first going unnoticed, but later developing into long-term swelling of the limbs. Because of this slow manifestation, many associated pathologic factors, such as long-term inflammation and tissue remodeling, including fibrosis, can develop prior to intervention [42]. Treatment is typically limited to compression therapy, although there have been some recent clinical trials with anti-inflammatory therapies and surgical interventions [34, 43]. Thus, lymphedema is a complex disease which in addition to the impairment of the lymphatic vessels themselves, includes impaired oxygen delivery due to greater diffusion distances, inflammation, changes in tissue architecture, and an altered physiological state of the interstitial fluid pressure gradients including elevated resistance to fluid flow [42, 44]. This warrants a holistic view of the pathophysiologic process to develop therapeutic approaches that can return tissues to their normal, optimized function. In this review, we explore the connection between lymphatic and microvascular networks, to promote a more thorough understanding of their normal physiological function and the relative contributions of microvessels and lymphatics to the perpetuation of lymphedema.

MICROVASCULAR LEAKAGE AND THE LYMPHATIC SAFETY FACTOR

Lymphedema can have genetic causes (primary lymphedema) or be secondary to injury or infection. For secondary lymphedema, the most common cause worldwide is filarial infections. However, in Western countries, the most common cause is lymph node biopsy or excision, typically performed as part of the treatment of breast cancer and other cancers with lymph node metastasis [18]. With both primary and secondary lymphedema, plasma colloid osmotic pressure is typically normal, however interstitial osmotic pressure is high, due to poor clearance of extravasated albumin by lymphatic vessels [45–47]. There is also a high amount of lipid content, reflecting impairment of another lymphatic function, clearance of high-density lipoproteins (HDL), also termed reverse cholesterol transport [48–50]. Lymphatic insufficiency can be diagnosed by evaluating lymphatic clearance with lymphoscintigraphy, and there are also newer experimental methods that utilize near-infrared imaging to view the degree of lymph flow in the limbs using subcutaneously injected tracer molecules [51–53].

While lymphatic insufficiency may be the main cause of lymphedema, it is currently unclear to what extent the chronic inflammatory conditions that arise in the pathogenesis of lymphedema contribute to sustaining the edema. In other words, in addition to the osmotic forces that sustain edema in the lymphedema setting, there may also be an inflammation-induced chronic elevation of microvascular permeability that augments the edema. A slightly different context is featured in the lymphedema distichiasis, caused by mutation of the FoxC2 gene. This primary lymphedema features lymphatic insufficiency due to impaired intraluminal valves in collecting lymphatics. However, FoxC2 mutations also impair venous valves [54], leading to an increase in capillary hydrostatic pressure, favoring increased filtration of plasma. Thus, in both contexts, the edema is caused by the combination of microvascular leakage and lymphatic insufficiency. Currently much effort is focused on the lymphatic aspects of lymphedema, however the potential role of ongoing, elevated microvascular leakage in perpetuating the tissue swelling in lymphedema has not been examined in detail.

This concept has been explored theoretically. In 1932, Krogh, Landis, and Taylor noted that a “margin of safety” against lymphedema must exist because venous pressure in limbs must be raised by 15–20 cm H₂O before large amounts of edematous fluid appears [55]. Guyton and colleagues provided a mathematical framework for this concept [56, 57]. They defined three edema safety factors in this framework, using the context of elevated capillary pressure, which would be expected to increase filtration (extravasation by convection). The first two safety factors are associated with the Starling forces and are: 1) the elevated interstitial hydrostatic pressure that occurs with filtration, which serves to limit additional filtration; and 2) the increased osmotic pressure gradient across the endothelium that occurs with filtration. For the latter, the magnitude of this increase in the osmotic gradient is much lower if permeability of the capillary or postcapillary venule to macromolecules such as albumin is high. The third safety factor was termed the lymphatic safety factor [44, 58], that considers the lymphatic capacity to clear leaked plasma. The lymphatic safety factor was considered to be as high as ten-fold greater than the typical rate of plasma leakage via the microcirculation into the tissues [58]. However, in cases in which the microvascular leakage of plasma exceeds the lymphatic drainage capacity, edema forms, and a vicious cycle of increased fluid leakage from microvessels can occur due to the increased osmotic gradient from the elevated plasma protein content in the interstitial space. Moreover, the recruitment of inflammatory cells in lymphedematous tissue and the activation of proinflammatory signaling pathways [46] may also exacerbate the microvascular leakage. Thus, a major question arises as to whether future therapies focused solely on restoring lymphatic insufficiency will be sufficient to mitigate lymphedema after it has formed.

Notably, recent clinical trials have explored potential benefits of anti-inflammatory drugs to ameliorate lymphedema. A completed phase 4 clinical trial showed some potential benefits from daily oral ketoprofen therapy, including a reduction in skin thickness and improved cutaneous architecture in affected limbs compared to placebo control [43]. This study also showed reduced numbers of cells with 5-lipoxygenase expression [43], in line with findings from a murine study suggesting that leukotriene-B4 (LTB4) inhibition can ameliorate experimental lymphedema [59]. An additional phase 2 clinical trial evaluating a protease inhibitor that reduces LTB4 levels, Ubenimex (ULTRA Trial, NCT02700529) recently concluded. While a full report has not yet been published, the study sponsor, Eiger BioPharmaceuticals, released a statement in 2018 that the primary endpoint, skin thickness, and the secondary endpoints, limb volume and bioimpedance, had not yet been met [60]. Another more recent trial published in 2021 introduced a treatment consisting of IL-4 and IL-13 neutralizing antibodies as a potential therapy that was able to improve patient quality of life as well as minimize several pathologic skin conditions including skin thickness, Collagen Type III deposition, and Th-2 cytokine presence [61]. Despite the mixed findings with anti-inflammatory approaches so far, these will likely be a key component to optimize future therapeutic protocols that more directly target lymphatic abnormalities that cause lymphatic insufficiency.

Because microvascular leakage of fluids and solutes is likely a continuous stress on lymphedematous tissues, an additional approach that could potentially be useful to reverse lymphedema could be to reduce microvascular permeability. The anti-inflammatory approaches may indirectly ameliorate microvascular hyperpermeability. However, more direct approaches could also be more effective. Several investigators have shown with both in vitro and animal models that the bioactive lipid sphingosine-1-phosphate (S1P) can reduce microvascular permeability in a variety of models of inflammation [62–66]. S1P and drugs that activate S1P receptor-1 have been shown to reduce microvascular leakage following experimental trauma in rodents [38, 40, 67]. This and other approaches to reduce plasma extravasation would likely produce some benefit as part of a therapeutic approach to resolve lymphedema.

CONSIDERING FLOW INTO INITIAL LYMPAHTIC VESSELS

Interstitial fluid is thought to predominately enter the blind ended initial lymphatic vessels, form lymph, and then flow through a network of pre-collectors and collecting lymphatics. Lymph flow can be categorized by four transient phases: 1) lymph formation; 2) transport in initial lymphatic networks; 3) transport within the collecting lymphatics; and 4) flow within lymph nodes. The following subsections will focus on the first phase and how fluid into initial lymphatics could be influenced by lymphatic endothelial cell junctions and the coordination between lymphatic and blood vessels. Consideration of lymph formation and the initial phase of flow in lymphatic capillaries, which have potential for remodeling as tissue demands change, prompts investigation of the overlaps between angiogenesis and lymphangiogenesis, direct connections between these two vascular systems, and new evidence for lymphatic/blood endothelial cell plasticity (Figs. 2, ​,33).

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Fig 2.

Cell Dynamics Involved in Angiogenesis and Lymphangiogenesis.

Capillary sprouting dynamics associated with blood and lymphatic vessel growth include cell differentiation, cell migration, cell recruitment, proliferation, growth factor production, responses to fluid flow, and local extracellular matrix manipulation. The common cell and molecular-level dynamics raise questions regarding their interdependence during microvascular remodeling. Printed with permission from Anita Impagliazzo.

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Fig 3.

Discoveries of Cell Dynamics That Motivate New Hypotheses Related to Lymphatic/Blood Vessel Interrelationships.

Discoveries demonstrating new dynamics relating angiogenesis and lymphangiogenesis include: 1) vascular island incorporation [99]; 2) vessel connections [92, 135]; and 3) vessel plasticity [91]. Each discovery supports the loss of lymphatic endothelial cell identity and raises new questions about functional effects. Printed with permission from Anita Impagliazzo.

Lymphatic Cell Identity and Plasticity

Examples of plasticity between blood and lymphatic vessel systems serve to frame how we view microvascular dynamics and tissue homeostasis. Physical connections between blood and lymphatic vessels can be caused changes in inflammatory cytokine levels, ECM, expression of genes controlling cell fate, and interactions with neighboring cell populations. Understanding these examples is critical to having a clear picture of what the microvascular lymphatic interface actually looks like, especially in pathological conditions. Lymphatic/blood vessel plasticity and its role in normal and pathological microvascular dynamics is a new, albeit controversial concept that challenges the established belief that the blood and lymphatic systems are two entirely distinct vascular systems. Our laboratories have introduced several examples detailing this phenomenon. We demonstrated that vascular islands, usually associated with lymphangiogenesis can reconnect with blood vessels during angiogenesis [96, 99, 100]. Time-lapse views of these islands also suggest that they can connect with lymphatic vessels. We have also observed lymphatic sprouts that lack Prox1, LYVE-1, and podoplanin [92]. Indicative of the loss of their lymphatic identity, these sprouts are also wrapped by NG2 positive pericytes. Most recently, we discovered that lymphatic vessels can form mosaic vessels with blood vessels [91]. The mosaic vessels are characterized by cells both positive and negative for lymphatic marker labeling (LYVE-1 and podoplanin). These examples, together with our discovery that nearby growth factor gradients and inflammatory cues can directly impact the development of blood/lymphatic connections in adult microvascular networks [92, 96, 101] highlight the need to understand how and why mosaic vessels can form.

The potential for lymphatic endothelial cell plasticity or reprogramming has been supported by multiple transgenic mouse studies where the loss of lymphatic identity and the formation of mutant blood-filled lymphatic vessels was observed. For example, deletion of Prox1 resulted in the formation of connections between blood and lymphatic vessels and caused lymphatic endothelial cells to transition to blood endothelial cells (Fig. 4) [102]. Other studies utilizing transgenic mice showed through intravenous injection of FITC-labeled dextran or BSI-lectin, that deletion of several other genes, including SLP-76 [103], Rac1 [104], O-glycan [105], and Fiaf [106] resulted in impaired blood-lymphatic separation and the formation of blood-lymphatic vessel connections during development. Collectively, this area of research highlights that lymphatic reprogramming can have functional implications. Finally, it should be noted that the concept of transdifferentiation is not totally new and could be bi-directional. For example, Cooley et al. reported human BECs in culture expressed altered gene profiles indicative of a lymphatic-like phenotype ability of dependent on the extracellular matrix environment [107, 108]. Adoption of a lymphatic phenotype might be caused by exposure to inflammatory cytokines, cell-cell interactions, cell-ECM interactions, and changes in the expression of key cell fate regulators [109].

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Fig 4.

Examples of Lymphatic/Blood Vessel Mispatterning.

Mispatterning in adult rat mesenteric microvascular networks can be characterized by observations of vessel segments with blood capillary like, non-lymphatic morphological or phenotypical signatures apparently connected with lymphatic vessels. A) Representative image of a remodeling microvascular network with multiple apparent blood-to-lymphatic vessel connections. PECAM labeling identifies blood and lymphatic vessels and LYVE-1 labeling is lymphatic-specific. Arrows identify locations of connections, which have previously been characterized by continuous sub-micron PECAM junctional labeling across vessel types [91, 92]. For this image, mesenteric networks were stimulated in culture to undergo angiogenesis and lymphangiogenesis. B) Representative image of a remodeling microvascular network with mosaic vessels characterized by apparent lymphatic segments along blood vessels. PECAM labeling identifies blood and lymphatic vessels and LYVE-1 labeling is lymphatic-specific. Arrows identify locations PECAM+, LYVE-1− segments, continuous with PECAM+, LYVE-1+ segments. The mosaic pattern indicates potential merging of lymphatic and non-lymphatic endothelial cells [91]. For this image, mesenteric networks were stimulated in culture to undergo angiogenesis and lymphangiogenesis. C) Representative image of a vessel segments with pericytes connected to lymphatic capillaries in an adult unstimulated microvascular network. PECAM labeling identifies lymphatic endothelial cells and NG2 labeling identifies vascular pericytes along vessels and nerves. Initial lymphatic vessels are supposed to be void of pericytes and the presence of NG2+ pericytes along segments characteristic of lymphatic sprouts raise questions about their origin and the lymphatic endothelial cell identity.

Beyond these physiological examples of connections at the microvascular lymphatic interface there are also developmental links that further show the interdependence of the two systems. Exploration into the non-venous origin of lymphatic vessels provides further examples of how new lymphatic vessels can form from previously unexpected sources, much like mosaic vessels could represent a new method of blood/lymphatic vessel transition of formation. Historical speculation on the interconnected nature of the blood and lymphatic systems started as early as 1902, when Sabin postulated that the primary lymphatic sacs originate from embryonic veins [110]. Resolution on the validity of this claim was later provided by studies utilizing transgenic Cre-Lox-based lineage tracing to assert that the origin of lymphatic endothelial progenitor cells, and thus the entire lymphatic system is solely venous in nature [111]. In some ways this paradigm supports the emergent evidence suggesting there is more to the story for lymphatic endothelial cell (LEC) identity. In recent years the origin of LECs has been challenged by observation of non-venous derived progenitor cells that give rise to lymphatic endothelial cells [112–114]. For example, different lineage tracing studies utilizing transgenic mice revealed and hemogenic endothelium and hemogenic endothelial progenitors as a source for part of the cardiac lymphatic network [113], for development of clusters of dermal lymphatic vasculature via lymphovasculogenesis [114], and for developing mesenteric lymphatic vasculature [115]. All these studies demonstrate heterogeneity in the origins of LEC progenitors in multiple different organ systems and thus raise questions about how these differences can results in distinct functional characterization especially in relation to nearby blood vasculature.

Discussion of the ability of macrophages and pericytes to influence and in some cases integrate with lymphatic vessels and cause lymphangiogenesis serve as a reminder of how plasticity in local cell populations beyond blood endothelium can affect the microvascular environment. These non-endothelial cell lineage cell types have been implicated as mediators of lymphangiogenesis and sources of lymphatic endothelial cells, further highlighting the potential and importance of cell plasticity in the microvascular lymphatic interface. For example, macrophages expressing the tyrosine kinase Syk have been shown to express lymphangiogenic and angiogenic factors including VEGF-C & VEGF-D [116]. In a different report, deletion of yolk sac-derived Cx3cr1+ tissue resident macrophages resulted in the formation of functional blood/lymphatic connections and disruption of lymphatic vessels in the heart [117]. Other studies have introduced specific markers, such as Toll-like Receptor 4 (TLR4), as having the capability to induce myeloid cells to undergo lymphatic transition [118]. In the inflamed cornea, CD11b+ macrophages have been identified as key lymphangiogenic contributors that can form lymphatic tube-like structures that express lymphatic markers [112]. Another study found that treating mice with lipopolysaccharide resulted in the rapid mobilization and incorporation of macrophage-derived LEC progenitors into inflammation-induced lymphatic vasculature [119]. Exploration of macrophages in both developmental and post-natal tissues have highlighted macrophages as supportive cell types that can secrete angiogenetic factors or transdifferentiate into LECs [69, 120]. Vascular pericytes are another understudied cell type defined as a peri-endothelial support cell along blood capillaries. Pericytes play a key role in endothelial cell proliferation, regulation of vessel diameter, and stabilization of blood vessels, particularly during angiogenic sprouting and vasculogenic tube formation [121]. One way that pericytes provide stability is by inducing the formation several of basement membrane components [122, 123]. In a mouse cornea neovascularization model (cornea is normally devoid of blood and lymphatic vasculature), transplanted bonemarrow derived pericyte precursors made up a small portion of LECs that incorporated into newly formed lymphatic structures [124]. This finding is particularly surprising, considering that lymphatic vessels have been shown to be devoid of pericytes [69] – a characteristic which could be related to lymphatic vessels also lacking a continuous basement membrane [125, 126]. All these cell players are highly interconnected and thus changes in any of their functional dynamics could have prominent effect on the tissue homeostasis.

FUTURE QUESTIONS AND CONCLUDING THOUGHTS

Does lymphangiogenesis necessarily lead to increased drainage? Where exactly do cells enter in the lymphatic system? What exactly is the impact of lymphatic structure on fluid flow? Are there preferential channels through which fluid flows from blood capillaries to initial lymphatic vessels? Are such channels tissue specific and what proportion of reabsorption might these channels account for in each tissue? Does microvascular hyperpermeability perpetuate lymphedema? These questions continue to drive lymphatic research and emphasize the importance of considering the integration of both lymphatic and blood systems in normal and pathological conditions (Table 1). In addition to reductionist studies focused on single molecular mechanisms, there is a need to understand the integration of function across the hierarchy of a lymphatic network and the coordination with blood vascular networks. This need for a holistic view is maybe most emphasized by considering that increased lymphangiogenesis is not always linked to improved lymph flow and a reduction in lymphedema [127]. For that matter, the resolution of lymphedema might not be dependent on lymphatic remodeling. Would decreasing permeability of the capillaries and venules reduce or alleviate lymphedema? This remains a significant question considering that specific therapeutics that reduce microvascular permeability by directly targeting blood endothelial cells, such as S1P [38, 40, 64] or sigma receptor agonists [128] remain in an experimental stage. Also, from a therapeutic perspective, there is concern that common agonists/drugs typically affect both vascular and lymphatic endothelium. For example, agents used to treat hypertension or angina, such as nifedipine, have also been shown to impair pump function in isolated lymphatic vessels [129, 130], and we have observed relaxation of isolated collecting lymphatics treated with the sigma receptor agonist Afobazole [131]. However, these effects may not necessarily occur in vivo when the drugs are administered orally or intravenously due to sponging by receptors on the blood endothelium, limiting how much drug reaches the lymphatic vessels. This is the case with nifedipine, which despite its profound impact on lymphatic vessels ex vivo, failed to cause significant alterations in lymphatic pumping after administration to humans in vivo [130].

Another question for development of potential therapeutics is whether differences in the ability to re-route lymph flow in different individuals could be a contributing factor for reversing lymphedema. Because many unidirectional vessels coalesce into to a main collector duct, an increase in resistance of this main collector, such as that with a lymphaticosclerosis, can potentially cause failure of the entire network [131, 132]. Redundancy in lymphatic networks prior to reaching outflow points is apparently protective against formation of breast cancer-related secondary lymphedema for people who have significant lymph drainage of the lateral arm through the Mascagni-Sappey pathway that bypasses the axillary lymph nodes [133]. The ongoing development of lymph node transplants as emerging therapy for lymphedema [34], and new tools for visualizing lymphatic flow in the surgical setting [134] offer promise for alleviating the bottleneck effect of damaged lymph trunks or lymph nodes that can significantly impair entire networks.

In summary, while lymphatic insufficiency is the primary cause of lymphedema, there are integrative problems with the local microcirculation and tissue inflammation that need to be considered. Successful future approaches to resolve lymphedema, in addition to addressing the direct cause of the lymphatic insufficiency, will likely also need ancillary treatment of inflammation and elevated microvascular leakage for optimal outcomes. Understanding the integrative nature of microvascular and lymphatic networks, including plasticity and the underlying genetic commonalities and differences between blood and lymphatic endothelial cells are key future areas of investigation.

ACKNOWLEDGEMENTS

Footnotes

REFERENCES