Microanatomy of the intestinal lymphatic system

The intestinal lymphatic system comprises two noncommunicating lymphatic networks: one containing the lacteals draining the villi and the connecting submucosal lymphatic network and one containing the lymphatics that drain the intestine muscular layer. These systems deliver lymph into a common network of collecting lymphatics originating near the mesenteric border. The intestinal lymphatic system serves vital functions in the regulation of tissue fluid homeostasis, immune surveillance, and the transport of nutrients; conversely, this system is affected by, and directly contributes to, disease processes within the intestine. Recent discoveries of specific lymphatic markers, factors promoting lymphangiogenesis, and factors selectively affecting the development of intestinal lymphatics, hold promise for unlocking the role of lymphatics in the pathogenesis of diseases affecting the intestine and for intestinal lymphatic selective therapies. Vital to progress in understanding how the intestinal lymphatic system functions is the integration of recent advances identifying molecular pathways for lymphatic growth and remodeling with advanced imaging modalities to observe lymphatic function and dysfunction in vivo.     

家兔盲肠和阑尾淋巴管的研究

用色素穿刺注入法、动脉内墨汁硝酸银水溶液注入法研究了家兔盲肠和阑尾淋巴管的起始、走向及所属淋巴结。盲肠和阑尾的毛细淋巴营均起始于固有膜,后依次穿过肠壁各层移行为系膜内淋巴管。后者经阑尾、盲肠淋巴结与肠系膜上淋巴结群中的相应淋巴结相联系。盲肠粘膜下层淋巴管内开始出现瓣膜;阑尾淋巴管瓣膜则在阑尾系膜缘内才出现。     

Structure of sinuses in the human lymph node

Summary A casting technique has been employed to display in three dimensions, the lymphatic microcirculation within the human lymph node. The casting compound filled the marginal sinus, and diffusely permeated the cortical lymphoid parenchyma. However, deep within the lymph node in the medullary region, the medium remained within the limits of the sinus walls. The casts showed well-defined channels appearing similar to vessels. These converged into larger vessels, which drained into efferent lymphatics leaving the node at the hilus.Electron microscopic examination showed that the outer wall of the marginal sinus and the trabecular side of trabecular sinuses had an intact, continuous endothelium with a basement membrane. However, gaps were present in the inner wall of the marginal sinus, as well as in the parenchymal wall of the trabecular sinus. In the medulla, the sinuses were lined by endothelial cells which appeared similar to macrophages. The sinus lining was incomplete and possessed numerous perforations. These observations indicated that sinus walls adjacent to connective tissue served as a barrier to cell movement, but those adjacent to a large lymphoid cell population had gaps, with cells in apparent transit between sinus lumen and parenchyma.     

A model for mechanics of primary lymphatic valves

Recent experimental evidence indicates that lymphatics have two valve systems, a set of primary valves in the wall of the endothelial cells of initial lymphatics and a secondary valve system in the lumen of the lymphatics. While the intralymphatic secondary valves are well described, no analysis of the primary valves is available. We propose a model for primary lymphatics valves at the junctions between lymphatic endothelial cells. The model consists of two overlapping endothelial extensions at a cell junction in the initial lymphatics. One cell extension is firmly attached to the adjacent connective tissue while the other cell extension is not attached to the interstitial collagen. It is free to bend into the lumen of the lymphatic when the lymphatic pressure falls below the adjacent interstitial fluid pressure. Thereby the cell junction opens a gap permitting entry of interstitial fluid into the lymphatic lumen. When the lymphatic fluid pressure rises above the adjacent interstitial fluid pressure, the endothelial extensions contact each other and the junction is closed preventing fluid reflow into the interstitial space. The model illustrates the mechanics of valve action and provides the first time a rational analysis of the mechanisms underlying fluid collection in the initial lymphatics and lymph transport in the microcirculation.     

Light and electron microscope observations of the lymphatic drainage units of the peritoneal cavity of rodents

Fluid, particles, and cells are taken up from the peritoneal cavity by lymphatic drainage units, which, in the mouse and rat, are located along the peritoneal surface of the muscular portion of the diaphragm. The drainage units are composed of three specifically differentiated components: a lymphatic lacuna, a covering of lacunar mesothelium, and intervening submesothelial connective tissue. The units are drained by connecting lymphatic vessels that cross the diaphragm to empty into collecting lymphatic vessels running along the pleural surface of the diaphragm. The collecting lymphatics empty into parasternal lymphatic trunks. In this report, we briefly review critical features of the drainage apparatus and describe new observations, summarized below, about their structure. Around the rim of stomata, the mesothelial openings that lead into the lymphatic lacunae, plasma membranes of lacunar mesothelial cells and of lacunar endothelial cells abut but are not linked to one another by recognizable junctional specializations. Lacunar endothelial cells often extend valve-like processes that bridge the distal end of the channel beneath the stoma. The configuration of the endothelial processes may be complex. Occasionally, processes from fibroblasts in the submesothelial connective tissue adjacent to stomata make contact with the interstitial surface of lacunar endothelial cells. A discontinuous elastic layer in the submesothelial connective tissue spans the roof of each lacuna. Connecting and collecting lymphatics, which drain lymphatic lacunae, possess endothelial valves. Possible functions for each of these newly described structural features are discussed.     

A tissue‐engineered model of the intestinal lacteal for evaluating lipid transport by lymphatics

Lacteals are the entry point of all dietary lipids into the circulation, yet little is known about the active regulation of lipid uptake by these lymphatic vessels, and there lacks in vitro models to study the lacteal—enterocyte interface. We describe an in vitro model of the human intestinal microenvironment containing differentiated Caco‐2 cells and lymphatic endothelial cells (LECs). We characterize the model for fatty acid, lipoprotein, albumin, and dextran transport, and compare to qualitative uptake of fatty acids into lacteals in vivo. We demonstrate relevant morphological features of both cell types and strongly polarized transport of fatty acid in the intestinal‐to‐lymphatic direction. We found much higher transport rates of lipid than of dextran or albumin across the lymphatic endothelial monolayer, suggesting most lipid transport is active and intracellular. This was confirmed with confocal imaging of Bodipy, a fluorescent fatty acid, along with transmission electron microscopy. Since our model recapitulates crucial aspects of the in vivo lymphatic–enterocyte interface, it is useful for studying the biology of lipid transport by lymphatics and as a tool for screening drugs and nanoparticles that target intestinal lymphatics. Biotechnol. Bioeng. 2009;103: 1224–1235. © 2009 Wiley Periodicals, Inc.     

Dual origin of avian lymphatics

The earliest signs of the lymphatic vascular system are the lymph sacs, which develop adjacent to specific embryonic veins. It has been suggested that sprouts from the lymph sacs form the complete lymphatic vascular system. We have studied the origin of the jugular lymph sacs (JLS), the dermal lymphatics and the lymph hearts of avian embryos. In day 6.5 embryos, the JLS is an endothelial-lined sinusoidal structure. The lymphatic endothelial cells (LECs) stain (in the quail) positive for QH1 antibody and soybean agglutinin. As early as day 4, the anlagen of the JLS can be recognized by their Prox1 expression. Prox1 is found in the jugular section of the cardinal veins, and in scattered cells located in the dermatomes along the cranio-caudal axis and in the splanchnopleura. In the quail, such cells are positive for Prox1 and QH1. In the jugular region, the veins co-express the angiopoietin receptor Tie2. Quail-chick-chimera studies show that the peripheral parts of the JLS form by integration of cells from the paraxial mesoderm. Intra-venous application of DiI-conjugated acetylated low-density lipoprotein into day 4 embryos suggests a venous origin of the deep parts of the JLS. Superficial lymphatics are directly derived from the dermatomes, as shown by dermatome grafting. The lymph hearts in the lumbo-sacral region develop from a plexus of Prox1-positive lymphatic capillaries. Both LECs and muscle cells of the lymph hearts are of somitic origin. In sum, avian lymphatics are of dual origin. The deep parts of the lymph sacs are derived from adjacent veins, the superficial parts of the JLS and the dermal lymphatics from local lymphangioblasts.     

Evidence for a second valve system in lymphatics: endothelial microvalves.

The mechanism for interstitial fluid uptake into the lymphatics remains speculative and unresolved. A system of intralymphatic valves exists that prevents reflow along the length of the lymphatic channels. However, these valves are not sufficient to provide unidirectional flow at the level of the initial lymphatics. We investigate here the hypothesis that initial lymphatics have a second, separate valve system that permits fluid to enter from the interstitium into the initial lymph channels but prevents escape back out into the tissue. The transport of fluorescent microspheres (0.31 microm) across endothelium of initial lymphatics in rat cremaster muscle was investigated with micropipette manipulation techniques. The results indicate that microspheres can readily pass from the interstitium across the endothelium into the lumen of the initial lymphatics. Once inside the lymphatic lumen, the microspheres cannot be forced out of the lumen even after elevation of the lymphatic pressure by outflow obstruction. Reaspiration of the microspheres inside the lymphatic lumen with a micropipette is blocked by the lymphatic endothelium. This blockade exists whether the aspiration is carried out at the microsphere entry site or anywhere along the initial lymphatics. Nevertheless, puncture of the initial lymphatic endothelium with the micropipette leads to rapid aspiration of intralymphatic microspheres. Investigation of lymphatic endothelial sections fixed during lymph pumping shows open interendothelial junctions not found in resting initial lymphatics. These results suggest that initial lymphatics have a (primary) valve system at the level of the endothelium. In conjunction with the classical (secondary) intralymphatic valves, the primary valves provide the mechanism that facilitates the unidirectional flow during periodic compression and expansion of initial lymphatics.     

The renal cortical lymphatic system in the rat, hamster, and rabbit

Rat, hamster, and rabbit renal cortical lymphatics were examined by light and electron microscopy. Rat and hamster kidneys possessed both intra- and interlobular lymphatics that were structurally similar at the light microscopic level. Ultrastructural examination of the hamster lymphatic endothelium, however, revealed an unusual arrangement of cytoplasmic extensions not seen in the other two species. The intralobular lymphatics were related primarily to tubules, afferent arterioles, and renal corpuscles and were consistent with lymph formation from both plasma filtrate and tubular reabsorbate. Interlobular lymphatics were seen in connective tissue associated with the interlobular blood vessels. Rabbit cortex contained only interlobular lymphatics. Cross-sectional area, maximum diameter, volume density, and profile density were determined by stereological measurements using a computer-based image analyzer. The morphological data from the rat were used, in combination with published values for lymph flow, to calculate the rate of lymph formation per unit area of endothelium in lymphatics of the renal cortex. Among kidneys fixed by retrograde perfusion, the cortical lymphatic system was most extensive in maximum diameter, volume density, and profile density. It was smallest in the rabbit and intermediate in the rat. Lower volume and profile density were found for rat kidneys fixed by the dripping technique. It was concluded that: tubular reabsorbate probably contributes to renal lymph in the rat and hamster, but not in the rabbit; significant differences exist in the extent of the renal lymphatic systems among the three species, with the hamster kidney having the richest network and the rabbit the poorest; the method of fixation influences the measured size and density of renal cortical lymphatics; and the estimated rate of lymph formation in the kidney of the rat is roughly comparable to that in the dog.     

Aging‐related anatomical and biochemical changes in lymphatic collectors impair lymph transport,fluid homeostasis,and pathogen clearance

The role of lymphatic vessels is to transport fluid, soluble molecules, and immune cells to the draining lymph nodes. Here, we analyze how the aging process affects the functionality of the lymphatic collectors and the dynamics of lymph flow. Ultrastructural, biochemical, and proteomic analysis indicates a loss of matrix proteins, and smooth muscle cells in aged collectors resulting in a decrease in contraction frequency, systolic lymph flow velocity, and pumping activity, as measured in vivo in lymphatic collectors. Functionally, this impairment also translated into a reduced ability for in vivo bacterial transport as determined by time‐lapse microscopy. Ultrastructural and proteomic analysis also indicates a decrease in the thickness of the endothelial cell glycocalyx and loss of gap junction proteins in aged lymph collectors. Redox proteomic analysis mapped an aging‐related increase in the glycation and carboxylation of lymphatic's endothelial cell and matrix proteins. Functionally, these modifications translate into apparent hyperpermeability of the lymphatics with pathogen escaping from the collectors into the surrounding tissue and a decreased ability to control tissue fluid homeostasis. Altogether, our data provide a mechanistic analysis of how the anatomical and biochemical changes, occurring in aged lymphatic vessels, compromise lymph flow, tissue fluid homeostasis, and pathogen transport.     

Distribution and fine structure of the lymphatic system in the human testis

Summary The distribution of lymph vessels in the human testis was investigated using ink injection methods, and light and electron microscopy. Lymph capillaries occur in the septula testis but are absent in the intertubular tissue. They consist of endothelial cells provided with an incomplete basal lamina and anchoring filaments of the adjacent connective tissue. Frequently, the endothelial cells are separated by gaps measuring up to 2m. The lymph capillaries of the septula testis are connected to lymph vessels in the rete testis and tunica albuginea. These vessels have occasional smooth muscle cells and valves. At the posterior margin of the testis, the network of lymph vessels merges into collecting ducts, which together with vessels derived from the rete testis are drained by the lymphatic system in the spermatic cord.Dedicated to Prof. Henriette Oboussier, Hamburg, on the occasion of her 65th birthday     

Therapeutic differentiation and maturation of lymphatic vessels after lymph node dissection and transplantation

Surgery or radiation therapy of metastatic cancer often damages lymph nodes, leading to secondary lymphedema. Here we show, using a newly established mouse model, that collecting lymphatic vessels can be regenerated and fused to lymph node transplants after lymph node removal. Treatment of lymph node-excised mice with adenovirally delivered vascular endothelial growth factor-C (VEGF-C) or VEGF-D induced robust growth of the lymphatic capillaries, which gradually underwent intrinsic remodeling, differentiation and maturation into functional collecting lymphatic vessels, including the formation of uniform endothelial cell-cell junctions and intraluminal valves. The vessels also reacquired pericyte contacts, which downregulated lymphatic capillary markers during vessel maturation. Growth factor therapy improved the outcome of lymph node transplantation, including functional reconstitution of the immunological barrier against tumor metastasis. These results show that growth factor-induced maturation of lymphatic vessels is possible in adult mice and provide a basis for future therapy of lymphedema.     

An investigation of lymphatic vessels in the feline dental pulp

The existence of lymphatic vessels in the dental pulp has been a matter of continuing controversy. We have now used light microscopy to examine semithin transverse sections of perfusion-fixed incisors and canines in cats. Lymphatics were found in all the teeth studied. In most teeth they were present in the coronal, middle, and apical regions of the pulp; but in a few they were lacking coronally and in the middle. Within individual teeth, lymphatics were found in the subodontoblastic zone or more centrally in the pulp; but none were found in the odontoblast layer or in the pulp horns. Vessels located by light microscopy were subsequently examined by transmission electron microscopy. Their ultrastructural features were typical of lymphatics and included irregular, attenuated endothelium with adjacent cells joined in different ways. Occasional gaps connected the extracellular spaces with their lumens, and abluminal endothelial projections appeared to form open end bulbs. There was very little basement membrane, but anchoring filaments were found near the abluminal surface of the endothelium and near collagen fibrils. The total cross-sectional area of lymphatic vessels was measured in semithin sections and, with pulp area, increased from the coronal region to the middle. However, both areas decreased from the middle to the apical region suggesting either that lymph flows faster as it reaches the foramens of the apical delta or that some vessels leave the tooth through lateral root canals. Using the methods of light and transmission electron microscopy, therefore, we have shown that pulp lymphatic vessels exist. Questions remain, however, about their distribution within teeth, variations between teeth, and routes of exit from teeth.     

Smooth muscle-endothelial cell communication activates Reelin signaling and regulates lymphatic vessel formation

Active lymph transport relies on smooth muscle cell (SMC) contractions around collecting lymphatic vessels, yet regulation of lymphatic vessel wall assembly and lymphatic pumping are poorly understood. Here, we identify Reelin, an extracellular matrix glycoprotein previously implicated in central nervous system development, as an important regulator of lymphatic vascular development. Reelin-deficient mice showed abnormal collecting lymphatic vessels, characterized by a reduced number of SMCs, abnormal expression of lymphatic capillary marker lymphatic vessel endothelial hyaluronan receptor 1 (LYVE-1), and impaired function. Furthermore, we show that SMC recruitment to lymphatic vessels stimulated release and proteolytic processing of endothelium-derived Reelin. Lymphatic endothelial cells in turn responded to Reelin by up-regulating monocyte chemotactic protein 1 (MCP1) expression, which suggests an autocrine mechanism for Reelin-mediated control of endothelial factor expression upstream of SMC recruitment. These results uncover a mechanism by which Reelin signaling is activated by communication between the two cell types of the collecting lymphatic vessels--smooth muscle and endothelial cells--and highlight a hitherto unrecognized and important function for SMCs in lymphatic vessel morphogenesis and function.     

Characteristics of Multi-Organ Lymphangiectasia Resulting from Temporal Deletion of Calcitonin Receptor-Like Receptor in Adult Mice

Adrenomedullin (AM) and its receptor complexes, calcitonin receptor-like receptor (Calcrl) and receptor activity modifying protein 2/3, are highly expressed in lymphatic endothelial cells and are required for embryonic lymphatic development. To determine the role of Calcrl in adulthood, we used an inducible Cre-loxP system to temporally and ubiquitously delete Calcrl in adult mice. Following tamoxifen injection, Calcrl^fl/fl/CAGGCre-ER™ mice rapidly developed corneal edema and inflammation that was preceded by and persistently associated with dilated corneoscleral lymphatics. Lacteals and submucosal lymphatic capillaries of the intestine were also dilated, while mesenteric collecting lymphatics failed to properly transport chyle after an acute Western Diet, culminating in chronic failure of Calcrl^fl/fl/CAGGCre-ER™ mice to gain weight. Dermal lymphatic capillaries were also dilated and chronic edema challenge confirmed significant and prolonged dermal lymphatic insufficiency. In vivo and in vitro imaging of lymphatics with either genetic or pharmacologic inhibition of AM signaling revealed markedly disorganized lymphatic junctional proteins ZO-1 and VE-cadherin. The maintenance of AM signaling during adulthood is required for preserving normal lymphatic permeability and function. Collectively, these studies reveal a spectrum of lymphatic defects in adult Calcrl^fl/fl/CAGGCre-ER™ mice that closely recapitulate the clinical symptoms of patients with corneal, intestinal and peripheral lymphangiectasia.     

Ultrastructural morphology of the canine thoracic duct and cisterna chyli

The ultrastructural morphology of the thoracic duct and cisterna chyli of the dog was examined using scanning and transmission electron microscopy. Examination of the cisterna chyli, reservoir of the lymphatic system, featured a number of afferent lymphatics draining into the cisterna: valves were however absent. The luminal surface of the endothelial lining of both the thoracic duct and cisterna demonstrated ovoid endothelial nuclei with numerous cellular ridges. Considerable variation existed in the number of smooth muscle cells lining the duct and cisterna in the contracted and distended state. Organelles and inclusions characteristic of endothelium and smooth muscle were identified. Reflux of lymph into the thoracic duct was prevented by a mono- and bicuspid valve situated at the lymphaticovenous junction.     

Comparison of lymphatic and venous interpositional autografts in experimental microsurgery of the canine lymphatics

In mongrel dogs, 56 autologous lymphatic and vein grafts were interpositioned to bridge a defect in the femoral collecting lymphatics. In one group, 26 lymphatic autografts were interpositioned with good results. No obstruction was observed over 6 months. In another group, 20 venous autografts were interpositioned after irrigation with heparinized saline and another 10 autografts were interpositioned without irrigation. After 1 week, four irrigated grafts were partially occluded with a red thrombus; after 6 months, all grafts were totally occluded. In a third group, 15 lymphaticolymphatic anastomoses were enveloped by a silicone sheet to provoke prolonged devascularization. None of the vessels was patent. Anastomotic patency was inspected in vivo postoperatively. The specimens were studied with light microscopy and scanning and transmission electron microscopy. Prolonged devascularization damaged the endothelial cells. The results show that the lymphatic vessel autograft is the best choice for an interpositional autografting to bridge a defect in lymphatic vessels.     

VEGF-D promotes the metastatic spread of tumor cells via the lymphatics

Metastasis to local lymph nodes via the lymphatic vessels is a common step in the spread of solid tumors. To investigate the molecular mechanisms underlying the spread of cancer by the lymphatics, we examined the ability of vascular endothelial growth factor (VEGF)-D, a ligand for the lymphatic growth factor receptor VEGFR-3/Flt-4, to induce formation of lymphatics in a mouse tumor model. Staining with markers specific for lymphatic endothelium demonstrated that VEGF-D induced the formation of lymphatics within tumors. Moreover, expression of VEGF-D in tumor cells led to spread of the tumor to lymph nodes, whereas expression of VEGF, an angiogenic growth factor which activates VEGFR-2 but not VEGFR-3, did not. VEGF-D also promoted tumor angiogenesis and growth. Lymphatic spread induced by VEGF-D could be blocked with an antibody specific for VEGF-D. This study demonstrates that lymphatics can be established in solid tumors and implicates VEGF family members in determining the route of metastatic spread.