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.     

Structure of lymphatic vessels in the rat thyroid gland

The lymphatic bed of the thyroid gland has been studied in 24 intact rats. Three techniques facilitating to reveal lymphatic vessels in the organ have been used: preparation of semithin sections, injection of the blood bed with methyl methacrylate with a successive chemical extraction of the preparations, injection of the blood bed with liquid solution of methyl methacrylate with a consecutive study of the preparations in the scanning electron microscope. Methods of electron histochemistry (revealing horseradish peroxidase) and kryofractography have been applied. Construction of the thyroid lymphatic bed, structure of the wall are described, fibroblastic membrane (F-membrane) is revealed and the signs are presented, that allow to differentiate F-membrane from endothelium of the lymphatic capillaries. The pathways of lymph outflow in the rat thyroid gland consist of the following links: interstitial space in the interlobular spaces of the gland, into them the tissue liquor (lymph) is filtered ; plates of the F-membrane regulating direction of excessive albumin-containing liquor in the lymphatic capillaries, surrounding groups of 5-11 follicles, embracing the microlobule of the gland and situating in the interlobular spaces, deferent lymphatic vessels.     

Topographic features of the organization of lymphatic capillaries and lipid resorption in the jejunal villi of the white rat

As demonstrate serial semithin sections and transmissive electron microscopy, there is not one but a group (2-7) of lymphatic capillaries with anastomoses between them in the villus of the white rat jejunum. In the superior parts of the villus the lumen in the lymphatic capillaries is maximal, and their distance to the epithelial basal membrane of the anterior and posterior surfaces is small. In the inferior part of the villus, when the size of the lumen in the lymphatic microvessels is minimal, the greatest distance between them and the basal membrane of epithelium covering the mentioned surfaces of the villus is noted. In the superior and middle parts of the villus paracellular transport of lipids from the interstitial space into the lumen of the lymphatic capillaries predominate, in the inferior part--transcellular is the main way of transport. The topographic peculiarities of the lymphatic microvessels in the superior and middle parts of the villus make, in combination with the active paracellular transport, the morphological basis of a more intensive absorbtion of lipids from the intestinal lumen.     

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.     

Multiscale modeling of lymphatic drainage from tissues using homogenization theory

Lymphatic capillary drainage of interstitial fluid under both steady-state and inflammatory conditions is important for tissue fluid balance, cancer metastasis, and immunity. Lymphatic drainage function is critically coupled to the fluid mechanical properties of the interstitium, yet this coupling is poorly understood. Here we sought to effectively model the lymphatic-interstitial fluid coupling and ask why the lymphatic capillary network often appears with roughly a hexagonal architecture. We use homogenization method, which allows tissue-scale lymph flow to be integrated with the microstructural details of the lymphatic capillaries, thus gaining insight into the functionality of lymphatic anatomy. We first describe flow in lymphatic capillaries using the Navier-Stokes equations and flow through the interstitium using Darcy's law. We then use multiscale homogenization to derive macroscale equations describing lymphatic drainage, with the mouse tail skin as a basis. We find that the limiting resistance for fluid drainage is that from the interstitium into the capillaries rather than within the capillaries. We also find that between hexagonal, square, and parallel tube configurations of lymphatic capillary networks, the hexagonal structure is the most efficient architecture for coupled interstitial and capillary fluid transport; that is, it clears the most interstitial fluid for a given network density and baseline interstitial fluid pressure. Thus, using homogenization theory, one can assess how vessel microstructure influences the macroscale fluid drainage by the lymphatics and demonstrate why the hexagonal network of dermal lymphatic capillaries is optimal for interstitial tissue fluid clearance.     

Adrenomedullin stabilizes the lymphatic endothelial barrier in vitro and in vivo

The lymphatic vascular system functions to maintain fluid homeostasis by removing fluid from the interstitial space and returning it to venous circulation. This process is dependent upon the maintenance and modulation of a semi-permeable barrier between lymphatic endothelial cells of the lymphatic capillaries. However, our understanding of the lymphatic endothelial barrier and the molecular mechanisms that govern its function remains limited. Adrenomedullin (AM) is a 52 amino acid secreted peptide which has a wide range of effects on cardiovascular physiology and is required for the normal development of the lymphatic vascular system. Here, we report that AM can also modulate lymphatic permeability in cultured dermal microlymphatic endothelial cells (HMVEC-dLy). AM stimulation caused a reorganization of the tight junction protein ZO-1 and the adherens protein VE-cadherin at the plasma membrane, effectively tightening the endothelial barrier. Stabilization of the lymphatic endothelial barrier by AM occurred independently of changes in junctional protein gene expression and AM^−/− endothelial cells showed no differences in the gene expression of junctional proteins compared to wildtype endothelial cells. Nevertheless, local administration of AM in the mouse tail decreased the rate of lymph uptake from the interstitial space into the lymphatic capillaries. Together, these data reveal a previously unrecognized role for AM in controlling lymphatic endothelial permeability and lymphatic flow through reorganization of junctional proteins.     

Histotopography and composition of the microcirculatory bed of the sciatic nerve in the rat

By means of macro-microscopical preparation methods, horseradish peroxidase injection, semithin sections, electron microscopy histotopography and composition of microvessels of the sciatic nerve have been studied in 20 mature male white rats. The perineural membrane has been stated to have no its own vascular network. In composition of the sciatic nerve and its large branches muscular venules are revealed; they run longitudinally along the whole length of the endoneural space and are tributaries of the epineural veins. A suggestion is made that presence of the contractile apparatus in the venular wall can play an important role in formation of the postcapillary resistance, regulating the hydrostatic pressure value in the lumen of endoneural capillaries ad intensity of liquor filtration into the interstitial space.     

The lymphatic system in body homeostasis: physiological conditions

The lymphatic system is an organized network composed of functionally interrelated lymphoid tissue, and transportation pathways of tissue fluid/lymph and lymphoid cells. Its main components are 1. migrating dendritic cells, macrophages and lymphocytes, organized lymphoid tissue such as lymph nodes, thymus, spleen, bone marrow, and lymphoid tissue in gut and lungs, liver lymphoid cells, and the dendritic cell network of nonlymphoid organs; 2. vessels (intercellular space, lymphatics, and perivascular spaces); 3. fluids (tissue fluid and lymph). The lymphatic system can be divided into the following compartments: peripheral (from the interstitial space to and within the nearest lymph node), and central (efferent lymphatics, cysterna chyli, and thoracic duct, all lymphoid organs). Organs and tissues with the most active afferent arm of the lymphatic system are skin, gut, and lungs. These are the body structures exposed to the external environment. All other nonlymphoid bodily tissues are also percolated by tissue fluid/lymph, and contain a network of dendritic cells and macrophages. Data obtained from normal human subjects on lymph composition and flow are presented. Future trends in lymphatic research are outlined.     

Engineered blood and lymphatic capillaries in 3-D VEGF-fibrin-collagen matrices with interstitial flow

In vitro endothelial cell organization into capillaries is a long standing challenge of tissue engineering. We recently showed the utility of low level interstitial flow in guiding the organization of endothelial cells through a 3-D fibrin matrix-containing covalently bound vascular endothelial growth factor (VEGF). Here this synergistic phenomenon was extended to explore the effects of matrix composition on in vitro capillary morphogenesis of human blood versus lymphatic endothelial cells (BECs and LECs). Different mixtures of fibrin and collagen were used in conjunction with constant concentrations of matrix-bound VEGF and slow interstitial flow over 10 days. Interestingly, the BECs and LECs each showed a distinct preference in terms of organization for matrix composition: LECs organized the most extensively in a fibrin-only matrix, while BEC organization was optimized in the compliant collagen-containing matrices. Furthermore, the BECs and LECs produced architecturally different structures; while BECs organized in thick, branched networks containing wide lumen, the LECs were elongated into slender, overlapping networks with fine lumen. These data demonstrate the importance of the 3-D matrix composition in facilitating and coordinating BEC and LEC capillary morphogenesis, which is important for in vitro vascularization of engineered tissues.     

Ultrastructure of mesenteric lymphatic postcapillaries of the small intestine of the human fetus

Lymphatic vessels of the small intestine mesentery have been investigated in 36 human fetuses 3-6-month-old. Lymphatic postcapillaries are the predominant link in the lymphomicrocirculatory bed studied. They differ from the capillaries only by presence of valves. Intersegmentary valves and velves at the places where the lymphatic capillaries get into the postcapillaries are described; they are situated along the whole length of the postcapillary. Both types of the valves can be uni- and besinusoid, and according to the number of endotheliocytes, covering the sinuses, uni- and multicellular. Cells of fibroblastic line and developing collagenous fibers are inevitable structural components and situate in their base. Ultrastructural peculiarities of the lymphatic postcapillary wall are described. Structural mechanisms responsible for dilatation and narrowing of the vascular lumen (formation of the pulse wave) are discussed. The data obtained support the conception successively elaborated by V. V. Kuprianov on the lymphatic postcapillary as an inevitable link of the lymphomicrocirculatory bed.     

A novel monoclonal antibody specific for lymphatic endothelium

The difficulty of identifying and differentiating lymphatic and blood microvessels in tissue sections can be overcome by a monoclonal antibody specific for lymphatic endothelium. Unfortunately, the only known antibody also reacts with the endothelium of some blood vessels. The technique of double immunization (passive, with an antiserum to blood endothelium, and active, with a suspension of lymphatic endothelial cells) was, therefore, used to increase the chances of recognizing specific lymphatic antigens by the mouse immune system. The monoclonal antibody obtained, LyMAb, a G₁ immunoglobulin, reacted strongly with the endothelium of bovine thoracic duct, mesenteric collecting vessels and lymphatic vessels of gall-bladder and lymph nodes and moderately with those of the intestinal wall. Blood vessels (intercostal arteries, azygos vein and blood microvessels of all organs tested) were consistently negative. The antibody was species-specific and did not react with formalin-fixed, paraffin-embedded sections. Cross-reactivity was limited to some connective tissue fibres and scattered cells in the lymph node parenchyma, intestinal villi and hepatic lobules.     

Anatomy of lymph nodes situated adjacent to the axilla in adult humans]

In 50 right and 50 left upper extremities examined in adult persons of both sex at the age of 28-90 years, delto-thoracic lymph nodes were revealed in 30% (right) and in 22% (left), and interthoracic lymph nodes--in 6% (right) and in 12% (left). The lymph nodes in question were revealed by the method of section after interstitial injection of Gerota's blue intradermally to fingers, palm, back of the hand deltoid area, lateral thoracic surface (at the level of the 6th intercostal space) and to the external part of the mammary gland. Injection was also performed into lymphatic vessels revealed by means of the interstitial injection. The delto-thoracic nodes were stated to situate in both the delto-thoracic sulcus and the delto-thoracic triangle. These vessels are situated along the course of the lateral collector of the free upper extremity. Deferent vessels of the delto-thoracic nodes flow into the apical axillary lymph nodes, into the deep and superficial cervical nodes, into the interthoracic lymph nodes and also into the subclavicular or into the jugular vein near a corresponding venous angle. Interthoracic lymph nodes, situated between musculus pectoralis major and minor, get their lymphatic vessels from lateral, inferior and central axillary nodes, from delto-thoracic nodes and also those lymphatic vessels that go from the mammary gland area. Deferent vessels of the interthoracic nodes flow into the apical axillary nodes.     

Visualisation and stereological assessment of blood and lymphatic vessels

The physiological processes involved in tissue development and regeneration also include the parallel formation of blood and lymphatic vessel circulations which involves their growth, maturation and remodelling. Both vascular systems are also frequently involved in the development and progression of pathological conditions in tissues and organs. The blood vascular system circulates oxygenated blood and nutrients at appropriate physiological levels for tissue survival, and efficiently removes all waste products including carbon dioxide. This continuous network consists of the heart, aorta, arteries, arterioles, capillaries, post-capillary venules, venules, veins and vena cava. This system exists in an interstitial environment together with the lymphatic vascular system, including lymph nodes, which aids maintenance of body fluid balance and immune surveillance. To understand the process of vascular development, vascular network stability, remodelling and/or regression in any research model under any experimental conditions, it is necessary to clearly and unequivocally identify and quantify all elements of the vascular network. By utilising stereological methods in combination with cellular markers for different vascular cell components, it is possible to estimate parameters such as surface density and surface area of blood vessels, length density and length of blood vessels as well as absolute vascular volume. This review examines the current strategies used to visualise blood vessels and lymphatic vessels in two- and three-dimensions and the basic principles of vascular stereology used to quantify vascular network parameters.     

Transport of exogenous albumin across lymphatic endothelium--an ultrastructural study

The pathways involved in protein transport across the lymphatic endothelium of the rat renal cortex after in vivo drip fixation were studied ultrastructurally. Both qualitative and quantitative analyses were made on tissues from physiological saline-injected rats and on tissues from rats injected intravenously with rat albumin (0.2 mg/g body wt., 0.4 mg/g body wt. or 0.8 mg/g body wt.). The volume density values of intracytoplasmic vesicles were as follows: (1) saline-injected rats: 0.024, (2) 0.2 mg/g body wt. albumin-injected rats: 0.029 (p less than 0.05), (3) 0.4 mg/g body wt. albumin-injected rats increased to 0.033 (p less than 0.01), (4) 0.8 mg/g body wt. albumin-injected rats had a value of 0.022. The numerical density of intracytoplasmic vesicles increased from 27/micrometers 3 after saline injection to 35/micrometers 3 (p less than 0.05) after 0.2 mg/g body wt. albumin injection. When rats were injected with 0.4 mg/g body wt. albumin, the numerical density was 44/micrometers 3 (p less than 0.01, in comparison with saline-injected rats) but this value decreased to 27/micrometers 3 in rats injected with 0.8 mg/g body wt. albumin. In the four groups, the range of vesicles was 28-38% opening into the lymphatic lumen and 7-16% of vesicles opening into the interstitial space. The remaining vesicles were free in the endothelial cytoplasm. There were more open junctions and wider cell junctions in the 0.8 mg/g body wt. albumin-injected group. It is concluded that normally albumin molecules are transported into the lymphatic capillaries by intracytoplasmic vesicles or through the normal interstitial space between endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

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.     

Comparison of flow rates and composition of ovarian lymph and blood in the day-16 pregnant rat

Rats (5) at Day 16 of pregnancy were anaesthetized and a modification of a venous outflow technique was used to collect ovarian venous blood and lymph for 2 h. Both fluids were analysed for progesterone, 20 alpha-dihydroprogesterone, total protein, transferrin and albumin concentrations. In addition SDS gel electrophoresis was carried out to obtain an initial indication of permeability of capillaries to the various protein fractions. The concentrations of progesterone and 20 alpha-dihydroprogesterone in ovarian lymph were only 37% and 48% respectively of the corresponding concentrations in the venous plasma. Total protein concentration in the lymph was 53% of the venous plasma. The albumin and transferrin concentrations were similarly lower in lymph than plasma but the difference was only significant for transferrin. This study confirms that the rate of lymph flow, per unit mass of tissue, is high for the ovary and represents about 1.1% of plasma flow. It shows also that of the total progestagens secreted only around 0.5% leave by the lymphatic route. The finding of relatively low progestagen concentrations in lymph questions the view that progestagens are transported by simple diffusion from the luteal cell to blood and raises the possibility of a counter-current flow between fluid in the interstitial space and blood.