Elastin Shapes Small Molecule Distribution in Lymph Node Conduits.

The spatial and temporal Ag distribution determines the subsequent T cell and B cell activation at the distinct anatomical locations in the lymph node (LN). It is well known that LN conduits facilitate small Ag distribution in the LN, but the mechanism of how Ags travel along LN conduits remains poorly understood. In C57BL/6J mice, using FITC as a fluorescent tracer to study lymph distribution in the LN, we found that FITC preferentially colocalized with LN capsule-associated (LNC) conduits. Images generated using a transmission electron microscope showed that LNC conduits are composed of solid collagen fibers and are wrapped with fibroblastic cells. Superresolution images revealed that high-intensity FITC is typically colocalized with elastin fibers inside the LNC conduits. Whereas tetramethylrhodamine isothiocyanate appears to enter LNC conduits as effectively as FITC, fluorescently-labeled Alexa-555-conjugated OVA labels significantly fewer LNC conduits. Importantly, injection of Alexa-555-conjugated OVA with LPS substantially increases OVA distribution along elastin fibers in LNC conduits, indicating immune stimulation is required for effective OVA traveling along elastin in LN conduits. Finally, elastin fibers preferentially surround lymphatic vessels in the skin and likely guide fluid flow to the lymphatic vessels. Our studies demonstrate that fluid or small molecules are preferentially colocalized with elastin fibers. Although the exact mechanism of how elastin fibers regulate Ag trafficking remains to be explored, our results suggest that elastin can be a potentially new target to direct Ag distribution in the LN during vaccine design.

Observation of the cytoarchitecture of the human esophageal mucosa with special attention to the lamina muscularis mucosae and the distribution of lymphatic vessels.

The cytoarchitecture of the esophageal mucosa was examined by using light microscopy, transmission electron microscopy, and scanning electron microscopy. The cytoarchitecture of the muscularis mucosae varied greatly among the cervical, thoracic, and abdominal esophagus, especially in the cervical esophagus, the muscularis mucosae suffered a loss and the distribution of lymphatic vessels also varied according to the site. It was suggested that these morphological differences would have a strong influence on the infiltration of esophageal cancer and the mode of lymph node metastasis.

Multiple mouse models of primary lymphedema exhibit distinct defects in lymphovenous valve development.

Lymph is returned to the blood circulation exclusively via four lymphovenous valves (LVVs). Despite their vital importance, the architecture and development of LVVs is poorly understood. We analyzed the formation of LVVs at the molecular and ultrastructural levels during mouse embryogenesis and identified three critical steps. First, LVV-forming endothelial cells (LVV-ECs) differentiate from PROX1(+) progenitors and delaminate from the luminal side of the veins. Second, LVV-ECs aggregate, align perpendicular to the direction of lymph flow and establish lympho-venous connections. Finally, LVVs mature with the recruitment of mural cells. LVV morphogenesis is disrupted in four different mouse models of primary lymphedema and the severity of LVV defects correlate with that of lymphedema. In summary, we have provided the first and the most comprehensive analysis of LVV development. Furthermore, our work suggests that aberrant LVVs contribute to lymphedema.

[THE STRUCTURE OF LYMPHATIC CAPILLARIES OF THE CILIARY BODY OF THE HUMAN EYE].

Using light microscopy, immunohistochemistry and electron microscopy, the structural organization of interstitial spaces and vessels of the ciliary body of the human eye (n = 5) were studied. The ciliary body was found to contain wide interstitial spaces--tissue clefts bound by collagen fibers and fibroblasts. Organ-specific lymphatic capillaries were also demonstrated in the ciliary body. According to the present findings and the lymphatic region concept, the first 2 elements of the lymphatic region of the eye were described: tissue clefts--prelymphatics and lymphatic capillaries of the ciliary body. The third element of the lymphatic region are the lymph nodes of the head and neck.

[Importance of lymphangiogenesis and ultrastructure of lymphatic capillaries in metastasis of malignant melanoma]. / Význam lymfangiogenézy a ultrastruktúry lymfatických kapilár pri metastázovaní malígneho melanómu.

Lymphangiogenesis - proliferation of lymphatic capillaries - in melanoma and in its vicinity plays an important role in metastatic process of malign melanoma cells in organism. Melanoma produces epidermal growth factor EGF which induces vascular endothelial growth factor VEGF-C and thereby starts lymphangiogenesis. It is very probable that malignant melanoma cells enter lymphatic capillaries also through specialized inter-endothelial junctions (endothelial microvalves, primary valves) situated in their walls. After entry of malign cells into lymphatic capillaries, these cells travel in lymph to the sentinel lymph node. Malign cells metastasize through lymphatic vessels probably also directly into distal regional lymphatic nodes. Metastasizing through blood vessels is suggestible also in early stage melanoma.

The Notch1-Dll4 signaling pathway regulates mouse postnatal lymphatic development.

The Notch signaling pathway plays a fundamental role during blood vessel development. Notch signaling regulates blood vessel morphogenesis by promoting arterial endothelial differentiation and providing spatial and temporal control over "tip cell" phenotype during angiogenic sprouting. Components of the Notch signaling pathway have emerged as potential regulators of lymphatic development, joining the increasing examples of blood vessel regulators that are also involved in lymphatic development. However, in mammals a role for the Notch signaling pathway during lymphatic development remains to be demonstrated. In this report, we show that blockade of Notch1 and Dll4, with specific function-blocking antibodies, results in defective postnatal lymphatic development in mice. Mechanistically, Notch1-Dll4 blockade is associated with down-regulation of EphrinB2 expression, been shown to be critically involved in VEGFR3/VEGFC signaling, resulting in reduced lymphangiogenic sprouting. In addition, Notch1-Dll4 blockade leads to compromised expression of distinct lymphatic markers and to dilation of collecting lymphatic vessels with reduced and disorganized mural cell coverage. Finally, Dll4-blockade impairs wound closure and severely affects lymphangiogenesis during the wound healing in adult mouse skin. Thus, our study demonstrates for the first time in a mammalian system that Notch1-Dll4 signaling pathway regulates postnatal lymphatic development and pathologic lymphangiogenesis.

Mast cells in common wolffish Anarhichas lupus L.: ontogeny, distribution and association with lymphatic vessels.

The morphology, ontogeny and tissue distribution of mast cells were studied in common wolffish(Anarhichas lupus L.) at the larval, juvenile and adult life stages using light and electron-microscopy and immunohistochemistry. Fish were sampled at 1 day, 1, 2, 3, 4, 8 and 12 weeks post-hatching in addition to 6 and 9 months and 2 years and older. From 8 weeks post-hatching, mast cells in common wolffish mainly appeared as oval or rounded cells 8-15 mm in diameter with an eccentrically placed, ovoid nucleus and filled with cytoplasmic granules up to 1.2 mm in diameter. Granules were refractile and eosinophilic to slightly basophilic in H&E and stained bright red with Martius-scarlet-blue and purple with pinacyanol erythrosinate in formalin-fixed tissues. Mast cells stained positive for piscidin 4 and Fc ε RI by immunohistochemistry. From 1 day to 4 weeks post-hatching, immature mast cell containing only a few irregularly sized cytoplasmic granules were observed by light and electron-microscopy in loose connective tissue of cranial areas. From 1 day post-hatching, these cells stained positive for piscidin 4 and Fc ε RI by immunohistochemistry. From 12 weeks post-hatching, mast cells showed a primarily perivascular distribution and were particularly closely associated with lymphatic vessels and sinuses. Mast cells were mainly located at the peripheral border of the adventitia of arteries and veins, while they were in intimate contact with the endothelium of the lymphatic vessels. Numerous mast cells were observed in the intestine. A stratum compactum, as described in salmonids, was not observed in wolffish intestine,nor were mast cells confined to a separate layer, a stratum granulosum. Lymphatic vessels consisting of endothelium, intimal connective tissue and a poorly developed basal lamina were observed in the intestine. Scanning electron microscopy was used to compare the structure and localization of intestinal mast cells of common wolffish and rainbow trout. Scanning electron microscopy also revealed endothelial surface features and confirmed the existence of three distinctly different types of vessels in the wolffish intestine. Rainbow trout mast cell granules appeared as intact globular structures while empty vacuoles were observed in common wolffish. Mast cells were closely associated with lymphatic vessels in common wolffish, but not in rainbow trout.

Structural relationship between microlymphatic and microvascullar blood vessels in the rabbit ventricular myocardium.

We investigated the distribution and relationship between draining lymphatic vessels, lymphatic capillaries, and microvascular blood vessels in rabbit ventricular tissue. The left and right ventricular tissue from 15 healthy adult rabbits was obtained, processed, and sectioned for analysis. 5'-nucleotidase-alkaline phosphatase (5'-Nase-Alpase) double staining was first used to identify lymphatic and blood vessels. Dual fluorescent immunohistochemical technique was then utilized with lymphatic endothelial cell marker podoplanin and blood vascular marker PAL-E. In addition, five ventricular samples were examined for ultrastructure using transmission electron microscopy (TEM). Draining lymphatic vessels and both lymphatic and blood capillaries were observed in the ventricular tissue. The lumens of draining lymphatic vessels were larger and irregular while the lymphatic capillaries were small in diameter and abundant. All lymphatic vessels were located among blood capillaries in the myocardium and aligned with the longitudinal axis of myocardial cells. The immunofluorescence double staining demonstrated that draining lymphatic vessels, lymphatic capillaries, and microvascular blood vessels were adjacent to each other and the cardiac myocyte with a ratio of lymphatic to microvascular blood vessels of approximately 1:1. This study suggests that lymphatic and blood capillaries exist in abundance and in nearly identical numbers in the ventricular myocardium and that they interweave with each other to comprise a complicated vessel network.

Magnetic resonance lymphography (MRL): point and counter-point.

Two preeminent lymphologists debate the findings, implications, interpretations, and value of magnetic resonance lymphography (MRL) in the evaluation of peripheral lymphedema. Their contrasting views are discussed in the context of different lymphatic imaging modalities including MRL, lymphoscintigraphy, and microscopic anatomy.

Xenotransplanted human prostate carcinoma (DU145) cells develop into carcinomas and cribriform carcinomas: ultrastructural aspects.

Androgen-independent, human prostate carcinoma cells (DU145) develop into solid, carcinomatous xenotransplants on the diaphragm of nu/nu mice. Tumors encompass at least two poorly differentiated cell types: a rapidly dividing, eosinophilic cell comprises the main cell population and a few, but large basophilic cells able to invade the peritoneal stroma, the muscular tissue, lymph vessels. Poor cell contacts, intracytoplasmic lumina, and signet cells are noted. Lysosomal activities are reflected by entoses and programmed cell deaths forming cribriform carcinomas. In large tumors, degraded cells may align with others to facilitate formation of blood supply routes. Malignant cells would spread via ascites and through lymphatics.

[Structure and function of lymphatic capillaries in synovial joint]. / K struktúre a funkcii lymfatických kapilár v synoviálnom klbe.

The review article focuses on the structure and function of lymphatic capillaries in connective tissues of skin, muscles and synovial membrane. Lymphatic capillaries (initial lymphatics) are formed from endothelial cells mutually arranged so that their intercellular junctions have different structure. In one of the different types of intercellular junctions the distal ends of endothelial cells overlap one another in the form of projections. Desmosomes are missing between the cell membranes of the internal and external projection without presence of any other junctional complexes. The external projection of the endothelial cell is tightly attached to the surrounding connective tissue with the help of anchoring filaments. The internal projection of the neighbouring endothelial cell may tilt over to the lumen of the lymphatic capillary and this may result in a several micrometers wide communication between the interstitium and the lumen with efflux of tissue fluid and leukocytes from the interstitium in to the lumen of the capillary. Lymphologists call the above mentioned openable intercellular junctions in their works also endothelial microvalves or primary valves. These primary valves in cooperation with classical (secondary) intralymphatic valves enable one way lymph flow during spontaneous contractions of the initial lymphatics. It is supposed that primary valves in lymphatic capillaries have an important role in the drainage of the connective tissues affected by inflammation also in the synovial joint.

Digging deeper into lymphatic vessel formation in vitro and in vivo.

BACKGROUND: Abnormal lymphatic vessel formation (lymphangiogenesis) is associated with different pathologies such as cancer, lymphedema, psoriasis and graft rejection. Lymphatic vasculature displays distinctive features than blood vasculature, and mechanisms underlying the formation of new lymphatic vessels during physiological and pathological processes are still poorly documented. Most studies on lymphatic vessel formation are focused on organism development rather than lymphangiogenic events occurring in adults. We have here studied lymphatic vessel formation in two in vivo models of pathological lymphangiogenesis (corneal assay and lymphangioma). These data have been confronted to those generated in the recently set up in vitro model of lymphatic ring assay. Ultrastructural analyses through Transmission Electron Microscopy (TEM) were performed to investigate tube morphogenesis, an important differentiating process observed during endothelial cell organization into capillary structures. RESULTS: In both in vivo models (lymphangiogenic corneal assay and lymphangioma), migrating lymphatic endothelial cells extended long processes exploring the neighboring environment and organized into cord-like structures. Signs of intense extracellular matrix remodeling were observed extracellularly and inside cytoplasmic vacuoles. The formation of intercellular spaces between endothelial cells led to tube formation. Proliferating lymphatic endothelial cells were detected both at the tips of sprouting capillaries and inside extending sprouts. The different steps of lymphangiogenesis observed in vivo are fully recapitulated in vitro, in the lymphatic ring assay and include: (1) endothelial cell alignment in cord like structure, (2) intracellular vacuole formation and (3) matrix degradation. CONCLUSIONS: In this study, we are providing evidence for lymphatic vessel formation through tunneling relying on extensive matrix remodeling, migration and alignment of sprouting endothelial cells into tubular structures. In addition, our data emphasize the suitability of the lymphatic ring assay to unravel mechanisms underlying lymphangiogenesis.

Endothelial cell O-glycan deficiency causes blood/lymphatic misconnections and consequent fatty liver disease in mice.

Mucin-type O-glycans (O-glycans) are highly expressed in vascular ECs. However, it is not known whether they are important for vascular development. To investigate the roles of EC O-glycans, we generated mice lacking T-synthase, a glycosyltransferase encoded by the gene C1galt1 that is critical for the biosynthesis of core 1-derived O-glycans, in ECs and hematopoietic cells (termed here EHC T-syn(-/-) mice). EHC T-syn(-/-) mice exhibited embryonic and neonatal lethality associated with disorganized and blood-filled lymphatic vessels. Bone marrow transplantation and EC C1galt1 transgene rescue demonstrated that lymphangiogenesis specifically requires EC O-glycans, and intestinal lymphatic microvessels in EHC T-syn(-/-) mice expressed a mosaic of blood and lymphatic EC markers. The level of O-glycoprotein podoplanin was significantly reduced in EHC T-syn(-/-) lymphatics, and podoplanin-deficient mice developed blood-filled lymphatics resembling EHC T-syn(-/-) defects. In addition, postnatal inactivation of C1galt1 caused blood/lymphatic vessel misconnections that were similar to the vascular defects in the EHC T-syn(-/-) mice. One consequence of eliminating T-synthase in ECs and hematopoietic cells was that the EHC T-syn(-/-) pups developed fatty liver disease, because of direct chylomicron deposition via misconnected portal vein and intestinal lymphatic systems. Our studies therefore demonstrate that EC O-glycans control the separation of blood and lymphatic vessels during embryonic and postnatal development, in part by regulating podoplanin expression.

Quantum dots trace lymphatic drainage from the mouse eye.

Glaucoma is a leading cause of blindness in the world, often associated with elevated eye pressure. Currently, all glaucoma treatments aim to lower eye pressure by improving fluid exit from the eye. We recently reported the presence of lymphatics in the human eye. The lymphatic circulation is known to drain fluid from organ tissues and, as such, lymphatics may also play a role in draining fluid from the eye. We investigated whether lymphatic drainage from the eye is present in mice by visualizing the trajectory of quantum dots once injected into the eye. Whole-body hyperspectral fluorescence imaging was performed in 17 live mice. In vivo imaging was conducted prior to injection, and 5, 20, 40 and 70 min, and 2, 6 and 24 h after injection. A quantum dot signal was observed in the left neck region at 6 h after tracer injection into the eye. Examination of immunofluorescence-labelled sections using confocal microscopy showed the presence of a quantum dot signal in the left submandibular lymph node. This is the first direct evidence of lymphatic drainage from the mouse eye. The use of quantum dots to image this lymphatic pathway in vivo is a novel tool to stimulate new treatments to reduce eye pressure and prevent blindness from glaucoma.

Immunohistochemical study of the lymphatic vessels in major salivary glands of the rat.

This study was designed to examine whether lymphatic vessels are present in the lobules of major salivary glands in the rat. Immunostaining with an antibody against podoplanin, a lymphatic endothelial cell marker, was performed on sections of the submandibular, sublingual and parotid glands. Light microscopy demonstrated podoplanin-positive lymphatic vessels around the interlobular ducts and the interlobular arteries and veins in the interlobular connective tissue in all of the major salivary glands. No podoplanin-positive lymphatic vessels were found in the lobules. Electron microscopy also demonstrated lymphatic endothelial cells showing podoplanin expression only in the interlobular connective tissue. These findings suggest that the lymphatic system of the rat major salivary glands originates in the interlobular connective tissue, and not in the lobules.

Evaluation of fluid channels in the human dura and superior sagittal sinus in older patients.

AIM: Recent studies suggest the leptomeninges may have a lymphatic drainage system connecting the subarachnoid space with dorsal cervical lymph nodes. The distribution and histologic features of any dural "lymphatics" has not been established or extensively studied. MATERIAL AND METHODS: Duras from 113 patients were evaluated including 96 formalin-fixed dural samples (mean age 62 years) collected from 2010 to 2015. An additional 17 samples were collected from Alzheimer's disease (AD) patients (mean age 81) autopsied between 1995 and 1997. Two, 2 cm length coronal sections were taken from mid-convexity dura, parallel to the middle meningeal artery, 3-5 cm below and perpendicular to the superior sagittal sinus (SSS). Sections of twenty-two cases were also taken of the SSS and peri-SSS dura. To screen for possible lymphatics, 52 dural and 22 SSS samples from these cases were evaluated with CD31 and podoplanin (D240) immunohistochemistry. RESULTS: Numerous unlined microscopic channels were found in 101 of 113 (89 %). In non-AD duras, 86 of 92 (93 %) had numerous channels. Duras with AD had significantly less channels i.e. 15 of 21(71 %, P = 0.048). None of the channels had lymphocytes, or neutrophils in their lumena. In the superior sagittal sinus, 9 of 9 non-AD and 12/13 AD SSS duras had fluid channels. Congo red stains revealed no amyloid-like material in the AD duras. Immunohistochemically, CD31 was not found in fluid channels but was in endothelium in 36 of 36 non-AD duras and in most blood vessels including 16 of 16 AD patients. Seven of 36 (19 %) with non-AD and 1 of 16 (6%) with AD had podoplanin in thin walled vessels suggestive of lymphatics but none showed staining in fluid channels. CONCLUSIONS: Unlined fluid channels are present in the dura but not clearly lymphatic.

Lymphatic marker podoplanin/D2-40 in human advanced cirrhotic liver--re-evaluations of microlymphatic abnormalities.

BACKGROUND: From the morphological appearance, it was impossible to distinguish terminal portal venules from small lymphatic vessels in the portal tract even using histochemical microscopic techniques. Recently, D2-40 was found to be expressed at a high level in lymphatic endothelial cells (LECs). This study was undertaken to elucidate hepatic lymphatic vessels during progression of cirrhosis by examining the expression of D2-40 in LECs. METHODS: Surgical wedge biopsy specimens were obtained from non-cirrhotic portions of human livers (normal control) and from cirrhotic livers (LC) (Child A-LC and Child C-LC). Immunohistochemical (IHC), Western blot, and immunoelectron microscopic studies were conducted using D2-40 as markers for lymphatic vessels, as well as CD34 for capillary blood vessels. RESULTS: Imunostaining of D2-40 produced a strong reaction in lymphatic vessels only, especially in Child C-LC. It was possible to distinguish the portal venules from the small lymphatic vessels using D-40. Immunoelectron microscopy revealed strong D2-40 expression along the luminal and abluminal portions of the cell membrane of LECs in Child C-LC tissue. CONCLUSION: It is possible to distinguish portal venules from small lymphatic vessels using D2-40 as marker. D2-40- labeling in lymphatic capillary endothelial cells is related to the degree of fibrosis in cirrhotic liver.

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.

Correlative 3D Imaging and Microfluidic Modelling of Human Pulmonary Lymphatics using Immunohistochemistry and High-resolution µCT.

Lung lymphatics maintain fluid homoeostasis by providing a drainage system that returns fluid, cells and metabolites to the circulatory system. The 3D structure of the human pulmonary lymphatic network is essential to lung function, but it is poorly characterised. Image-based 3D mathematical modelling of pulmonary lymphatic microfluidics has been limited by the lack of accurate and representative image geometries. This is due to the microstructural similarity of the lymphatics to the blood vessel network, the lack of lymphatic-specific biomarkers, the technical limitations associated with image resolution in 3D, and sectioning artefacts present in 2D techniques. We present a method that combines lymphatic specific (D240 antibody) immunohistochemistry (IHC), optimised high-resolution X-ray microfocus computed tomography (µCT) and finite-element mathematical modelling to assess the function of human peripheral lung tissue. The initial results identify lymphatic heterogeneity within and between lung tissue. Lymphatic vessel volume fraction and fractal dimension significantly decreases away from the lung pleural surface (p < 0.001, n = 25 and p < 0.01, n = 20, respectively). Microfluidic modelling successfully shows that in lung tissue the fluid derived from the blood vessels drains through the interstitium into the lymphatic vessel network and this drainage is different in the subpleural space compared to the intralobular space. When comparing lung tissue from health and disease, human pulmonary lymphatics were significantly different across five morphometric measures used in this study (p ≤ 0.0001). This proof of principle study establishes a new engineering technology and workflow for further studies of pulmonary lymphatics and demonstrates for the first time the combination of correlative µCT and IHC to enable 3D mathematical modelling of human lung microfluidics at micrometre resolution.

Valves Are a Conserved Feature of the Zebrafish Lymphatic System.

The lymphatic system comprises blind-ended tubes that collect interstitial fluid and return it to the circulatory system. In mammals, unidirectional lymphatic flow is driven by muscle contraction working in conjunction with valves. Accordingly, defective lymphatic valve morphogenesis results in backflow leading to edema. In fish species, studies dating to the 18th century failed to identify lymphatic valves, a precedent that currently persists, raising the question of whether the zebrafish could be used to study the development of these structures. Here, we provide functional and morphological evidence of valves in the zebrafish lymphatic system. Electron microscopy revealed valve ultrastructure similar to mammals, while live imaging using transgenic lines identified the developmental origins of lymphatic valve progenitors. Zebrafish embryos bearing mutations in genes required for mammalian valve morphogenesis show defective lymphatic valve formation and edema. Together, our observations provide a foundation from which to further investigate lymphatic valve formation in zebrafish.