Plasticity of button-like junctions in the endothelium of airway lymphatics in development and inflammation.

Endothelial cells of initial lymphatics have discontinuous button-like junctions (buttons), unlike continuous zipper-like junctions (zippers) of collecting lymphatics and blood vessels. Buttons are thought to act as primary valves for fluid and cell entry into lymphatics. To learn when and how buttons form during development and whether they change in disease, we examined the appearance of buttons in mouse embryos and their plasticity in sustained inflammation. We found that endothelial cells of lymph sacs at embryonic day (E)12.5 and tracheal lymphatics at E16.5 were joined by zippers, not buttons. However, zippers in initial lymphatics decreased rapidly just before birth, as buttons appeared. The proportion of buttons increased from only 6% at E17.5 and 12% at E18.5 to 35% at birth, 50% at postnatal day (P)7, 90% at P28, and 100% at P70. In inflammation, zippers replaced buttons in airway lymphatics at 14 and 28 days after Mycoplasma pulmonis infection of the respiratory tract. The change in lymphatic junctions was reversed by dexamethasone but not by inhibition of vascular endothelial growth factor receptor-3 signaling by antibody mF4-31C1. Dexamethasone also promoted button formation during early postnatal development through a direct effect involving glucocorticoid receptor phosphorylation in lymphatic endothelial cells. These findings demonstrate the plasticity of intercellular junctions in lymphatics during development and inflammation and show that button formation can be promoted by glucocorticoid receptor signaling in lymphatic endothelial cells.

The specific effect of 2-methoxyestradiol on lymphatic vascular endothelial cells.

Lymphatic metastasis of tumors is one of the most important prognostic factors and provides valuable information for decisions on appropriate surgical protocols. Recent studies have demonstrated that lymphangiogenesis of lymphatic vascular endothelial cells into tumors is a key event in lymphatic metastasis. Therefore, control of lymphangiogenesis is a promising strategy for treatment or prevention of tumor metastasis and lymphatic disorders. However, mechanisms of lymphangiogenesis or its specific inhibition are not well-understood. In this study we examined effects of various types of signaling inhibitors on tube formation in human lymphatic microvascular endothelial cells (LECs) and blood microvascular endothelial cells (BECs) in vitro. We found that tube formation of LECs was specifically inhibited by 2-methoxyestradiol (2ME). This observation is of potential benefit in understanding the molecular mechanism of lymphangiogenesis. Furthermore, 2ME could therefore offer specific protection against lymphatic metastasis and lymphangiogenesis-related diseases.

Influence of IFN- alpha and IFN- gamma on lymphangiogenesis.

Malignant cancers commonly spread by local invasion followed by metastasis through venous or lymphatic passages or both to distant sites. Angiogenesis and its relation to tumor growth and metastasis have been extensively researched. To date, however, the role played by lymphangiogenesis and metastasis of cancer has been overlooked. Inhibition of lymphangiogenesis, compared with inhibition of angiogenesis, may provide new insight to the mechanisms of metastasis of cancers. The current study was designed to examine the effect of two commonly used inhibitors of angiogenesis, interferon-alpha (IFN-alpha ) and IFN-gamma, on the growth and proliferation of lymphatic endothelial (LE) cells isolated from pig thoracic duct under in vitro condition. The LE cells were isolated and marked using specific markers, such as VEGFR-3 and LYVE-1, before experimental studies. The results showed that treatment of LE cells derived from the thoracic duct with these two inhibitors caused a decrease in the rate of cell proliferation in a dose-dependent manner, as assessed by MTT assays (tetrazolium salt colorimetric assay). Cell migration rate was assessed by the speed at which the cell migrated out from the scrape-wound margin; the speed of migration of LE cells was significantly inhibited in a dose-dependent fashion compared with controls. Treatment with both IFN-alpha and IFN-gamma caused an increase in apoptosis of LE cells, as assessed by Hoechst staining and caspase-3 staining. Our results showed that both IFN-alpha and IFN-gamma were able to inhibit LE cell growth in a dose-dependent manner and that the inhibition may be through induction of apoptosis of endothelial cells.

Postnatal development of lymphatic vessels and their smooth muscle cells in the rat diaphragm: a confocal microscopic study.

This paper reports on how lymphatic vessels and their smooth muscle cells develop in the diaphragm of postnatal rats. Lymphatic endothelial cells in the diaphragm were labeled by an intraperitoneal injection of DiI-labeled acetylated low-density lipoprotein (DiIac-LDL). During postnatal week 1, DiI-ac-LDL was detected in many free cells in addition to distinct endothelial cells that formed lymphatic vessels. Occasionally, saccular lymphatics isolated from previously formed lymphatics were recognized; these were referred to as lymphatic islands. The DiI-ac-LDL-labeled free and lymphatic endothelial cells showed immunoreactivity for CD 34 and Flt-4, but most of them did not express either OX 62 or ED 1 immunoreactivity, with only some showing ED 1 immunoreactivity. This suggests that most of the DiI-ac-LDL-labeled elements were lymphatic endothelial cells, and that some were macrophages. After postnatal week 1, the DiI-ac-LDL positive cells were restricted to lymphatic vessels. Until postnatal week 6, lymphatic vessels increased as the diaphragm enlarged. Towards the end of postnatal week 2, free cells expressing alpha-smooth muscle actin (alpha-SMA) immunoreactivity increased in the diaphragm, and some of these were in contact with lymphatics. A coarse plexus of smooth muscle cells surrounding the lymphatic vessels first appeared at postnatal week 2, and this plexus became denser with age. Our findings indicate that lymphatic vessels are formed not only by sprouting from previously formed lymphatic vessels but also by migrating endothelial cells, and that smooth muscle cells may be differentiated from mesenchymal cells to form a plexus surrounding the lymphatic vessels.

Peritoneal lymphatic stomata of the diaphragm in the mouse: process of their formation.

BACKGROUND: Lymphatic stomata are channels connecting the peritoneal cavity with the lymphatics in the diaphragm. The process of sequential formation of the stomata has not been studied. The objective of this study was to examine the morphogenesis of the lymphatic stomata in mice. METHODS: Ultrathin sections of diaphragms from ddY mice obtained on embryonic day 18 and postnatal days 0, 4, and 10 were observed with a transmission electron microscope. RESULTS: By embryonic day 18 and postnatal day 0, lymphatics were already observed in the submesothelial connective tissue on the peritoneal side of the fetal diaphragm. The lymphatic endothelial cells, but not the mesothelial cells covering the diaphragm, protruded short cytoplasmic processes into the submesothelial connective tissue, and these almost reached the basal surfaces of individual mesothelial cells. By postnatal days 4 and 10, the lymphatic endothelial cells frequently protruded cytoplasmic processes into the submesothelial connective tissue, and the endothelial cell processes broke the continuity of both the basal lamina beneath the mesothelial cells and the submesothelial connective tissue. Neighboring endothelial processes formed a pair of U-shaped folds that were connected with each other via intercellular junctions at the apexes of the U-shaped folds. The disassembly of the intercellular junctions between the U-shaped folds was observed, and the basal surface of the mesothelial cell faced the lymphatic lumen. Dehiscence of the intercellular junctions between the mesothelial cells overlaying the lymphatics was observed, and lymphatic stomata were present. On the pleural side of the diaphragm, lymphatics were already present on embryonic day 18, but it was not observed that the endothelial process spanned the submesothelial connective tissue to the basal surface of the mesothelial cell. CONCLUSIONS: These results suggest the following process of the formation of the lymphatic stomata. (1) Neighboring lymphatic endothelial cells span the submesothelial connective tissue to the basal surfaces of mesothelial cells. (2) The lymphatic stomata are formed by the disassembly of the intercellular junctions between the neighboring endothelial cells and between the mesothelial cells overlying the endothelial cells.

Lymphangiogenesis in vitro: formation of lymphatic capillary-like channels from confluent monolayers of lymphatic endothelial cells.

Lymphatic endothelial cells grown long term in culture form lymphatic capillary-like tubes. Examination by light and transmission electron microscopy showed that these structures were closed loops composed of one to several cells connected by intercellular junction to form a luminal space. This first demonstration of lymphangiogenesis in confluent monolayer cultures of lymphatic endothelial cells (a) showed that collagen type I accelerated lymphatic capillary tube formation, whereas fibronectin and matrigel had no effect; b) provided a model to study lymphatic endothelial cell function and differentiation; and c) offered a possibility to distinguish differences between the process of lymphangiogenesis and angiogenesis by testing various factors and conditions that effect endothelial cell behavior.

Development of the high endothelial venule in rat lymph node autografts.

Vascular reconstruction during rat lymph node regeneration was investigated in autotransplanted mesenteric lymph node fragments, which had been implanted in the renal parenchyma. In addition to light microscopy, vascular casting and transmission electron microscopy were used. From day 3 onwards capillaries grew into the autografts together with lymphatic vessels. The capillaries showed obvious signs of proliferation by day 5. The surviving interstitial cells at the outer border of the transplant produced extracellular substance. High endothelial venules (HEV) differentiated from capillaries from about day 7. A first sign of their development was a vessel with a narrow, branching luminal space and with endothelial cells containing rich cytoplasm and small Golgi complexes. As the Golgi complexes grew and the cisternae and vesicles increased, the lumen dilated, the cell coat on the luminal surface became prominent, and, finally, lymphocytes emigrated through these venules from around day 10. The typical lymph node structure was complete by day 28. These results suggest that the interaction among the remaining interstitial cells, invading capillaries, and lymphatic penetration results in differentiation and maturation of HEV in lymph node regeneration. The development of Golgi complexes is strongly associated with lymphocyte emigration from the blood.

Lymphatic endothelium isolation, characterization and long-term culture.

Using a collagenase trypsin-EDTA treatment, we have been able to successfully isolate and grow primary cultures of the lymphatic endothelium (LEC) that were subcultured, frozen for storage, subsequently thawed with good recovery and growth, and serially subcultured. The morphological features of cultured LEC were consistent with that observed for the endothelium of intact lymphatic vessels. A prominent feature of growing cultures was the appearance of large vacuoles in the perinuclear region of the cytoplasm, which became filled with fluid and cell debris engulfed from the culture medium. The basal cell surface lacked a well defined basal lamina and anchoring filaments were observed extending from the basal plasmalemmal surface into the underlying substratum. LEC in cultures were also positive for Factor VIII-related antigen. However, specific granules, characteristic of Weibel-Palade bodies were not observed in ultrathin sections of confluent cultures. F-actin was identified in LEC cultures using fluorescein phalloidin, and in confluent cultures actin filaments were located at the periphery of the cell as a continuous circumferential thin band and short filamentous bundles in the central part of the cell. By using heparin and endothelial cell growth supplement in the culture medium we have been able to grow stable cultures of lymphatic endothelial cells that could be maintained when serially subcultured for over two years. These LEC cultures provide an in vitro model for investigating the function and biochemical properties of the lymphatic endothelium.

Developmental regulation of vascular addressin expression: a possible role for site-associated environments.

Tissue selective traffic of lymphocytes into different lymphoid organs is mediated by adherence of blood borne lymphocytes to specialized endothelial cells lining the high endothelial venules (HEV) in lymphoid organs. Lymphocytes discriminate between HEV in peripheral lymph nodes and in mucosal lymphoid tissues by means of membrane associated lymphocyte homing receptors adhering to their putative HEV ligands, the vascular addressins. The expression of particular vascular addressins on HEV is site- or tissue-selective and may be directed by factors unique to a specific location or lymph node environment. In this study we investigated the impact of regional environments on lymph node HEV differentiation and function. Experimentally, this problem was approached by the transplantation of lymph nodes from one region to a second region. The sites selected for receipt of tissues were the mesentery, a mucosal site, and the popliteal fossa, a peripheral site. We found that the phenotype of lymph node HEV following transplantation was influenced by both donor age and transplantation site. The transplantation site could influence vascular addressin expression, when tissues were obtained from late fetal or early neonatal donors and not when obtained from adult donors. Transplanted adult tissues retained their pre-transplantation vascular addressin expression phenotype regardless of transplantation site. Thus the endothelium within adult lymph nodes may be committed to expression of a particular addressin or addressins during lymph node development. It is also possible that regulatory cells or structures present within lymph nodes at the time of transplantation direct vascular addressin expression following tissue engraftment.(ABSTRACT TRUNCATED AT 250 WORDS)