A Tale of Two Models: Mouse and Zebrafish as Complementary Models for Lymphatic Studies

Lymphatic vessels provide essential roles in maintaining fluid homeostasis and lipid absorption. Dysfunctions of the lymphatic vessels lead to debilitating pathological conditions, collectively known as lymphedema. In addition, lymphatic vessels are a critical moderator for the onset and progression of diverse human diseases including metastatic cancer and obesity. Despite their clinical importance, there is no currently effective pharmacological therapy to regulate functions of lymphatic vessels. Recent efforts to manipulate the Vascular Endothelial Growth Factor-C (VEGFC) pathway, which is arguably the most important signaling pathway regulating lymphatic endothelial cells, to alleviate lymphedema yielded largely mixed results, necessitating identification of new targetable signaling pathways for therapeutic intervention for lymphedema. Zebrafish, a relatively new model system to investigate lymphatic biology, appears to be an ideal model to identify novel therapeutic targets for lymphatic biology. In this review, we will provide an overview of our current understanding of the lymphatic vessels in vertebrates, and discuss zebrafish as a promising in vivo model to study lymphatic vessels.     

Regulation of lymphangiogenesis--from cell fate determination to vessel remodeling

Lymphatic vessels are important for the maintenance of normal tissue fluid balance, immune surveillance and adsorption of digested fats. During the past decade, the identification of lymphatic-specific markers and growth factors has enabled detailed studies of the lymphatic system, and gain- and loss-of-function experiments have greatly increased our understanding of the mechanisms of normal lymphatic development. Understanding the basic biology has provided novel insights into the pathologic conditions of the lymphatic system that contribute to lymphedema, inflammation or lymphatic metastasis, and opened possibilities for the development of better therapeutic strategies. Here we review the current knowledge about the molecular mechanisms regulating the development of the lymphatic vasculature; of the differentiation of lymphatic endothelial cells, of the regulation of the growth of lymphatic vessels, and of remodeling of the vasculature into a network consisting of lymphatic capillaries and collecting lymphatic vessels. Furthermore, we will discuss the molecular mechanisms involved in the pathological conditions of the lymphatic vessels.     

Intestinal and peri-tumoral lymphatic endothelial cells are resistant to radiation-induced apoptosis

Radiation therapy is a widely used cancer treatment, but it is unable to completely block cancer metastasis. The lymphatic vasculature serves as the primary route for metastatic spread, but little is known about how lymphatic endothelial cells respond to radiation. Here, we show that lymphatic endothelial cells in the small intestine and peri-tumor areas are highly resistant to radiation injury, while blood vessel endothelial cells in the small intestine are relatively sensitive. Our results suggest the need for alternative therapeutic modalities that can block lymphatic endothelial cell survival, and thus disrupt the integrity of lymphatic vessels in peri-tumor areas.     

Endolymphatic infusion: human surgical investigation and application

The ability to influence changes within the lymphatic system, and to exert a therapeutic influence on the composition and quality of the lymph, may have a substantial impact on the therapeutic outcome in disease interventions. This report concerns our experience with endolymphatic infusion (ELI) of drugs, an approach that we have utilized in a surgical context to destroy bacteria within lymphatic vessels and lymph nodes. Experimentally, we have demonstrated that a variety of antibiotics will temporarily decrease the contractile activity of the lymphatic vessels and reduce the volume of lymph flow. Based on our initial observations, we have applied endolymphatic infusion to clinical care. ELI was evaluated in the treatment of 330 patients, utilizing protocols to alter the characteristics of lymph flow.     

Interaction between the extracellular matrix and lymphatics: Consequences for lymphangiogenesis and lymphatic function

The lymphatic system is important for body fluid balance as well as immunological surveillance. Due to the identification of new molecular markers during the last decade, there has been a recent dramatic increase in our knowledge on the molecular mechanisms involved in lymphatic vessel growth (lymphangiogenesis) and lymphatic function. Here we review data showing that although it is often overlooked, the extracellular matrix plays an important role in the generation of new lymphatic vessels as a response to physiological and pathological stimuli. Extracellular matrix-lymphatic interactions as well as biophysical characteristics of the stroma have consequences for tumor formation, growth and metastasis. During the recent years, anti-lymphangiogenesis has emerged as an additional therapeutic modality to the clinically applied anti-angiogenesis strategy. Oppositely, enhancement of lymphangiogenesis in situations of lymph accumulation is seen as a promising strategy to a set of conditions where few therapeutic avenues are available. Knowledge on the interaction between the extracellular matrix and the lymphatics may enhance our understanding of the underlying mechanisms and may ultimately lead to better therapies for conditions where reduced or increased lymphatic function is the therapeutic target.     

Use of a whole-slide imaging system to assess the presence and alteration of lymphatic vessels in joint sections of arthritic mice

Abstract

We investigated the presence and alteration of lymphatic vessels in joints of arthritic mice using a whole-slide imaging system. Joints and long bone sections were cut from paraffin blocks of two mouse models of arthritis: meniscal-ligamentous injury (MLI)-induced osteoarthritis (OA) and TNF transgene (TNF-Tg)-induced rheumatoid arthritis (RA). MLI-OA mice were fed a high fat diet to accelerate OA development. TNF-Tg mice were treated with lymphatic growth factor VEGF-C virus to stimulate lymphangiogenesis. Sections were double immunofluorescence stained with anti-podoplanin and alpha-smooth muscle actin antibodies. The area and number of lymphatic capillaries and mature lymphatic vessels were determined using a whole-slide imaging system and its associated software. Lymphatic vessels in joints were distributed in soft tissues mainly around the joint capsule, ligaments, fat pads and muscles. In long bones, enriched lymphatic vessels were present in the periosteal areas adjacent to the blood vessels. Occasionally, lymphatic vessels were observed in the cortical bone. Increased lymphatic capillaries, but decreased mature lymphatic vessels, were detected in both OA and RA joints. VEGF-C treatment increased lymphatic capillary and mature vessel formation in RA joints. Our findings suggest that the lymphatic system may play an important role in arthritis pathogenesis and treatment.     

Lymphatic smooth muscle: the motor unit of lymph drainage

Embedded into the wall of collecting lymphatic vessels and trunks, the lymphatic smooth muscles are cardinal to the functions of the lymphatic system. Their intrinsic contractile property--the intrinsic lymph pump--through rhythmical and phasic contractions of the vessels, represents the principal mechanism by which lymph flow is generated. Through changes in tonic constrictions, lymphatic smooth muscles also modulate lymph flow resistance. Lymphatic smooth muscles are sensitive to physical and chemical stimuli, mediating changes in their activity and modulating lymphatic drainage. Because lymphatic smooth muscles play such an important role in fluid transport, their dysfunction may be a component of many inflammatory disease states. This review presents recent findings on the physiology and cellular biology of lymphatic smooth muscles and discusses the importance of these cells for the function of the lymphatic system in physiological and pathophysiological situations.     

Abnormal lymphatic vessel development in neuropilin 2 mutant mice

Neuropilin 2 is a receptor for class III semaphorins and for certain members of the vascular endothelial growth factor family. Targeted inactivation of the neuropilin 2 gene (Nrp2) has previously shown its role in neural development. We report that neuropilin 2 expression in the vascular system is restricted to veins and lymphatic vessels. Homozygous Nrp2 mutants show absence or severe reduction of small lymphatic vessels and capillaries during development. This correlated with a reduction of DNA synthesis in the lymphatic endothelial cells of the mutants. Arteries, veins and larger, collecting lymphatic vessels developed normally, suggesting that neuropilin 2 is selectively required for the formation of small lymphatic vessels and capillaries.     

The lymphatic vasculature in disease

Blood vessels form a closed circulatory system, whereas lymphatic vessels form a one-way conduit for tissue fluid and leukocytes. In most vertebrates, the main function of lymphatic vessels is to collect excess protein-rich fluid that has extravasated from blood vessels and transport it back into the blood circulation. Lymphatic vessels have an important immune surveillance function, as they import various antigens and activated antigen-presenting cells into the lymph nodes and export immune effector cells and humoral response factors into the blood circulation. Defects in lymphatic function can lead to lymph accumulation in tissues, dampened immune responses, connective tissue and fat accumulation, and tissue swelling known as lymphedema. This review highlights the most recent developments in lymphatic biology and how the lymphatic system contributes to the pathogenesis of various diseases involving immune and inflammatory responses and its role in disseminating tumor cells.     

Endothelial-epithelial communication in polycystic kidney disease: Role of vascular endothelial growth factor signalling

Whereas targeting the cyst epithelium and its molecular machinery has been the prevailing clinical strategy for polycystic kidney disease, the endothelium, including blood vasculature and lymphatics, is emerging as an important player in this disorder. In this Review, we provide an overview of the structural and functional alterations to blood vasculature and lymphatic vessels in the polycystic kidney. We also discuss evidence for vascular endothelial growth factor signalling, otherwise critical for endothelial cell development and maintenance, as being a fundamental molecular pathway in polycystic kidney disease and a potential therapeutic target for modulating cyst expansion.     

Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis

Metastasis of breast cancer occurs primarily through the lymphatic system, and the extent of lymph node involvement is a key prognostic factor for the disease. Whereas the significance of angiogenesis for tumor progression has been well documented, the ability of tumor cells to induce the growth of lymphatic vessels (lymphangiogenesis) and the presence of intratumoral lymphatic vessels have been controversial. Using a novel marker for lymphatic endothelium, LYVE-1, we demonstrate here the occurrence of intratumoral lymphangiogenesis within human breast cancers after orthotopic transplantation onto nude mice. Vascular endothelial growth factor (VEGF)-C overexpression in breast cancer cells potently increased intratumoral lymphangiogenesis, resulting in significantly enhanced metastasis to regional lymph nodes and to lungs. The degree of tumor lymphangiogenesis was highly correlated with the extent of lymph node and lung metastases. These results establish the occurrence and biological significance of intratumoral lymphangiogenesis in breast cancer and identify VEGF-C as a molecular link between tumor lymphangiogenesis and metastasis.     

A fully coupled fluid-structure interaction model of the secondary lymphatic valve

Abstract

The secondary lymphatic valve is a bi-leaflet structure frequent throughout collecting vessels that serves to prevent retrograde flow of lymph. Despite its vital function in lymph flow and apparent importance in disease development, the lymphatic valve and its associated fluid dynamics have been largely understudied. The goal of this work was to construct a physiologically relevant computational model of an idealized rat mesenteric lymphatic valve using fully coupled fluid-structure interactions to investigate the relationship between three-dimensional flow patterns and stress/deformation within the valve leaflets. The minimum valve resistance to flow, which has been shown to be an important parameter in effective lymphatic pumping, was computed as 268?g/mm⁴?s. Hysteretic behavior of the lymphatic valve was confirmed by comparing resistance values for a given transvalvular pressure drop during opening and closing. Furthermore, eddy structures were present within the sinus adjacent to the valve leaflets in what appear to be areas of vortical flow; the eddy structures were characterized by non-zero velocity values (up to ～4?mm/s) in response to an applied unsteady transvalvular pressure. These modeling capabilities present a useful platform for investigating the complex interplay between soft tissue motion and fluid dynamics of lymphatic valves and contribute to the breadth of knowledge regarding the importance of biomechanics in lymphatic system function.     

Hyaluronan promotes tumor lymphangiogenesis and intralymphantic tumor growth in xenografts

Hyaluronan （HA）, a high molecular weight glycosaminoglycan in the extracellular matrix, has been implicated in the promotion of malignant phenotypes, including tumor angiogenesis. However, little is known about the effect of HA on tumor-associated lymphangiogenesis. In this study, mouse hepatocellular carcinoma Hca-F cells combined with or without HA were injected subcutaneously into C3H/Hej mice, then angiogenesis and lymphangiogenesis of implanted tumors were examined by immunostaining for plateletendothelial cell adhesion molecule-1 and lymphatic vascular endothelial hyaluronan receptor-1 respectively. Interestingly, we found HA promotes tumor lymphangiogenesis and the occurrence of intratumoral lymphatic vessels, but has little effect on tumor angiogenesis. Moreover, HA also promotes intralymphatic tumor growth, although it is not sufficient to potentiate lymphatic metastasis. These results suggest that HA, which is elevated in most malignant tumor stroma, may also play a role in tumor progression by promoting lymphangiogenesis.     

Local lymphogenic migration pathway in normal mouse spleen

Although the immunological and hemodynamical significance of the spleen is of great importance, few reports detail the lymphatic vessels in this organ. We have used an immunohistochemical three-dimensional imaging technique to characterize lymphatic vessels in the normal mouse spleen and have successfully demonstrated their spatial relationship to the blood vascular system for the first time. Lymphatic markers, such as LYVE-1, VEGFR-3, and podoplanin, show different staining patterns depending on their location in the spleen. LYVE-1-positive lymphatic vessels run reverse to the arterial blood flow along the central arteries in the white pulp and trabecular arteries and exit the spleen from the hilum. These lymphatic vessels are surrounded by type IV collagen, indicating that they are collecting lymphatic vessels rather than lymphatic capillaries. Podoplanin is expressed not only in lymphatic vessels, but also in stromal cells in the white pulp. These podoplanin-positive cells form fine meshworks surrounding the lymphatic vessels and central arteries. Following intravenous transplantation of lymphocytes positive for green fluorescent protein (GFP⁺) into normal recipient mice, donor cells appear in the meshworks within 1 h and accumulate in the lymphatic vessels within 6 h after injection. The GFP⁺ cells further accumulate in a draining celiac lymph node through the efferent lymphatic vessels from the hilum. These meshworks might therefore act as an extravascular lymphatic pathway and, together with ordinary lymphatic vessels, play a primary role in the cell traffic of the spleen, additional to the blood circulatory system.     

Intralobular Pulmonary Lymphatic Distribution in Normal Human Lung Using D2-40 Antipodoplanin Immunostaining

It has been assumed for a long time that except for limited areas close to respiratory bronchioles or their satellite arteries, there is no evidence of lymphatic vessels deep in the pulmonary lobule. An immunohistochemical study using the D2-40 monoclonal antibody was performed on normal pulmonary samples obtained from surgical specimens, with particular attention to the intralobular distribution of lymphatic vessels. This study demonstrated the presence of lymphatics not only in the connective tissue surrounding the respiratory bronchioles but also associated with intralobular arterioles and/or small veins even less than 50 μm in diameter. A few interlobular lymphatic vessels with a diameter ranging from 10 μm to 20 μm were also observed further away, in interalveolar walls. In conclusion, this study, using the D2-40 monoclonal antibody, demonstrated the presence of small lymphatic channels within the normal human pulmonary lobules, emerging from interalveolar interstitium, and around small blood vessels constituting the paraalveolar lymphatics. This thin intralobular lymphatic network may play a key pathophysiological role in a wide variety of alveolar and interstitial lung diseases and requires further investigation. (J Histochem Cytochem 57:643–648, 2009)     

Lymphatic function and responses in periodontal disease

Extravasated fluid, proteins and cells are returned into the circulation by lymphatic vessels that are also important in immune cell trafficking. Lymphatic vessels in gingiva are located in lamina propria, and traverse the external surface of the alveolar bone. Lack of gingival lymphatics has been shown to increase the interstitial fluid pressure and fluid volume, thus showing that lymphatics are important for fluid drainage also in this tissue. Gingival lymphatic vessels require continuous signaling by the growth factors VEGF-C and D via their receptor VEGFR-3 for their maintenance, factors that are expressed in the gingival epithelium and also in immune cells in lamina propria. VEGF-C seems to be of critical importance for lymphangiogeneses induced during periodontal disease development. Mice are protected against periodontitis by lymphatics clearing bacteria and bacterial products and promoting humoral immune responses. CCL21, a ligand important for dendritic cell migration, has been found to be downregulated in lymphatics from patients with periodontitis. Such patients may have impaired gingival lymphatic function due to high enzymatic activity and thus loss of structural components in the interstitium. At present there are few studies on the role of lymphatic vessels in periodontal disease making this a rather unexplored field.     

Intralobular lymphatic vessels and their relationship to blood vessels in the mouse thymus

Summary The spatial distribution and fine structure of the lymphatic vessels within the thymic lobules of normal and hydrocortisone-injected mice were studied by light- and electron microscopy. The lymphatic vessels of the cortex and medulla of normal thymus are irregularly shaped spaces closely associated with branches of the intralobular artery and vein. The overall distribution of these vessels in the greatly involuted thymus of hydrocortisone-treated mice is essentially the same as in the normal thymus. The wall of the lymphatic vessels consists of only a layer of endothelial cells supported by underlying reticular cells. The luminal surface of the endothelial cell is smooth, but trabecular processes are often seen. There are three morphological types of intercellular contacts between contiguous cells, namely, end-to-end, overlapping and interdigitating. The lymphatic vessel has anchoring filaments and collagen fibrils, but a basal lamina is either absent, or if present, is discontinuous. This is in contrast to the continuous basal lamina of the venule. The perivascular space surrounding the postcapillary venule opens into a terminal lymphatic vessel at the cortico-medullary junction and in the medulla. Lymphocytes are seen penetrating the lymphatic endothelium, particularly in acutely involuted thymuses. These findings suggest that the intralobular lymphatic vessels may originate from the vacuities that surround the postcapillary venules, and the lymphatic system may function as a pathway for the migration of lymphocytes into or out of the lymphatic circulation.     

The structure of lymphatic capillaries in lymph formation.

The lymphatic vascular system consists of endothelial lined vessels which begin as blind-end tubes or saccules that are located within the connective tissue areas. This system serves as a one-way drainage apparatus for the removal of diffusible substances as well as plasma proteins that escape the blood capillaries. If permitted to accumulate, these escaped components would deplete the circulatory system of its plasma colloids and disrupt the balance of forces responsible for the control of fluid movement and the exchange of gases and fluids across the blood vascular wall. The lymphatic capillaries are strategically placed and anatomically constructed to permit a continuous and rapid removal of the transient interstitial fluids, plasma proteins, and cells from the interstitium. Structurally the lymphatic capillaries consist of a continuous endothelium that is extremely attenuated over major aspects of its diameter, except in the perinuclear region which bulges into the lumen. These vessels lack a continuous basal lamina and maintain a close relationship with the adjoining interstitium by way of anchoring filaments. The adjacent cells are extensively overlapped and lack adhesion devices in many areas. When electron-opaque tracers are injected intravenously (i.e., horseradish peroxidase and ferritin), subsequent electron microscopic examination of tissues reveals the presence of tracer particles within the interstitium and the lymphatic capillary lumen. These particles gain access into the lymphatic capillaries via two major pathways: 1) the intercellular clefts of patent junctions and 2) plasmalemmal vesicles (pinocytotic vesicles). Another salient feature of the lymphatic endothelial cell includes the presence of numerous cytoplasmic filaments, which are similar in morphology to the actin filaments observed in a variety of cell types. The ultrastructural features of the lymphatic capillaries are discussed in relation to their role in the removal of interstitial fluids and particulate matter, and in the formation of lymph.     

Lymphatic pump function in the inflamed gut

The role of the lymphatic circulation to actively remove fluid, cells, proteins, and other particles from the interstitium to prevent mounting edema is well appreciated, but whether and how this function is compromised during inflammation has been scarcely investigated. We discuss here the mechanisms of lymphatic pumping and their modulation in inflammatory conditions or by inflammatory mediators in the context of inflammatory bowel disease (IBD), an ensemble of disorders typically described with abnormal or dysfunctional intestinal or mesenteric lymphatic vessels. We report our findings showing impaired mesenteric lymphatic contractile activity in an animal model of intestinal inflammation that recapitulates some features of IBD and suggests a role for prostanoids in this dysfunction. With the knowledge that prostaglandin E(2) and prostacyclin are implicated in IBD pathogenesis and induce a potent inhibition of lymphatic pumping, we established the pharmacological profile for these prostaglandin receptors in mesenteric lymphatic vessels and their respective role in pumping inhibition. Inhibition of mesenteric lymphatic pumping during inflammation may be a cause of edema, compromised immune response, and granuloma associated with IBD.     

Probing the effect of morphology on lymphatic valve dynamic function

The lymphatic system is vital to the circulatory and immune systems, performing a range of important functions such as transport of interstitial fluid, fatty acid, and immune cells. Lymphatic vessels are composed of contractile walls and lymphatic valves, allowing them to pump lymph against adverse pressure gradients and to prevent backflow. Despite the importance of the lymphatic system, the contribution of mechanical and geometric changes of lymphatic valves and vessels in pathologies of lymphatic dysfunction, such as lymphedema, is not well understood. We develop a fully coupled fluid–solid, three-dimensional computational model to interrogate the various parameters thought to influence valve behavior and the consequences of these changes to overall lymphatic function. A lattice Boltzmann model is used to simulate the lymph, while a lattice spring model is used to model the mechanics of lymphatic valves. Lymphatic valve functions such as enabling lymph flow and preventing backflow under varied lymphatic valve geometries and mechanical properties are investigated to provide an understanding of the function of lymphatic vessels and valves. The simulations indicate that lymphatic valve function is optimized when valves are of low aspect ratio and bending stiffness, so long as these parameters are maintained at high enough values to allow for proper valve closing. This suggests that valve stiffening could have a profound effect on overall lymphatic pumping performance. Furthermore, dynamic valve simulations showed that this model captures the delayed response of lymphatic valves to dynamic flow conditions, which is an essential feature of valve operation. Thus, our model enhances our understanding of how lymphatic pathologies, specifically those exhibiting abnormal valve morphologies such as has been suggested to occur in cases of primary lymphedema, can lead to lymphatic dysfunctions.