Somatic activating mutations in PIK3CA cause generalized lymphatic anomaly.

Generalized lymphatic anomaly (GLA) is a vascular disorder characterized by diffuse or multifocal lymphatic malformations (LMs). The etiology of GLA is poorly understood. We identified four distinct somatic PIK3CA variants (Glu542Lys, Gln546Lys, His1047Arg, and His1047Leu) in tissue samples from five out of nine patients with GLA. These same PIK3CA variants occur in PIK3CA-related overgrowth spectrum and cause hyperactivation of the PI3K-AKT-mTOR pathway. We found that the mTOR inhibitor, rapamycin, prevented lymphatic hyperplasia and dysfunction in mice that expressed an active form of PIK3CA (His1047Arg) in their lymphatics. We also found that rapamycin reduced pain in patients with GLA. In conclusion, we report that somatic activating PIK3CA mutations can cause GLA, and we provide preclinical and clinical evidence to support the use of rapamycin for the treatment of this disabling and deadly disease.

Hdac3 regulates lymphovenous and lymphatic valve formation.

Lymphedema, the most common lymphatic anomaly, involves defective lymphatic valve development; yet the epigenetic modifiers underlying lymphatic valve morphogenesis remain elusive. Here, we showed that during mouse development, the histone-modifying enzyme histone deacetylase 3 (Hdac3) regulates the formation of both lymphovenous valves, which maintain the separation of the blood and lymphatic vascular systems, and the lymphatic valves. Endothelium-specific ablation of Hdac3 in mice led to blood-filled lymphatic vessels, edema, defective lymphovenous valve morphogenesis, improper lymphatic drainage, defective lymphatic valve maturation, and complete lethality. Hdac3-deficient lymphovenous valves and lymphatic vessels exhibited reduced expression of the transcription factor Gata2 and its target genes. In response to oscillatory shear stress, the transcription factors Tal1, Gata2, and Ets1/2 physically interacted with and recruited Hdac3 to the evolutionarily conserved E-box-GATA-ETS composite element of a Gata2 intragenic enhancer. In turn, Hdac3 recruited histone acetyltransferase Ep300 to form an enhanceosome complex that promoted Gata2 expression. Together, these results identify Hdac3 as a key epigenetic modifier that maintains blood-lymph separation and integrates both extrinsic forces and intrinsic cues to regulate lymphatic valve development.

Angiopoietin-Tie signalling in the cardiovascular and lymphatic systems.

Endothelial cells that form the inner layer of blood and lymphatic vessels are important regulators of vascular functions and centrally involved in the pathogenesis of vascular diseases. In addition to the vascular endothelial growth factor (VEGF) receptor pathway, the angiopoietin (Ang)-Tie system is a second endothelial cell specific ligand-receptor signalling system necessary for embryonic cardiovascular and lymphatic development. The Ang-Tie system also regulates postnatal angiogenesis, vessel remodelling, vascular permeability and inflammation to maintain vascular homoeostasis in adult physiology. This system is implicated in numerous diseases where the vasculature has an important contribution, such as cancer, sepsis, diabetes, atherosclerosis and ocular diseases. Furthermore, mutations in the TIE2 signalling pathway cause defects in vascular morphogenesis, resulting in venous malformations and primary congenital glaucoma. Here, we review recent advances in the understanding of the Ang-Tie signalling system, including cross-talk with the vascular endothelial protein tyrosine phosphatase (VE-PTP) and the integrin cell adhesion receptors, focusing on the Ang-Tie system in vascular development and pathogenesis of vascular diseases.

Sphingosine-1-phosphate lyase is expressed by CD68+ cells on the parenchymal side of marginal reticular cells in human lymph nodes.

Lymph nodes (LNs) form the intersection between the vascular and lymphatic systems. Lymphocytes and antigen-presenting cells (APCs) traffic between these systems, but the barriers crossed during this trafficking in human LNs are poorly defined. We identified a population of cells in human LNs that lines the boundary between the parenchyma and lymphatic sinuses, consistent with descriptions of marginal reticular cells (MRCs) in murine LNs. Human MRCs are CD141(high) podoplanin(+), CD90(+), ICAM1(+), and VCAM1(+) but lack endothelial and hematopoietic cell markers, or alpha-smooth muscle actin. We then examined expression of the enzyme sphingosine-1-phosphate (S1P) lyase (SGPL1) relative to the boundary defined by MRCs. SGPL1 expression was almost exclusively restricted to cells on the parenchymal side of MRCs, consistent with a role in maintaining the S1P gradient between the sinuses and the parenchyma. Surprisingly the cells expressing SGPL1 in the parenchyma were CD68(+) APCs. CD68(+) APCs generated from human monocytes were able to internalize and irreversibly degrade S1P, and this activity was inhibited by the S1P analogue FTY720. This work provides a map of the key structures at the boundary where human lymphocytes egress into sinuses, and identifies a novel potential mechanism for the activity of S1P analogues in humans.

Lymphatic diamine oxidase secretion stimulated by fat absorption is linked with histamine release.

Diamine oxidase (DAO) is abundantly expressed in mammalian small intestine catalyzing the oxidative breakdown of polyamines and histamine. The aim of this study was to determine the relationship between stimulation of intestinal diamine oxidase secretion with intestinal fat absorption and histamine release. Conscious intestinal lymph fistula rats were used. The mesenteric lymph ducts were cannulated and intraduodenal tubes were installed for the infusion of Liposyn II 20% (an intralipid emulsion). Lymphatic DAO activity and protein secretion were analyzed by radiometric assay and Western blot, respectively. Lymphatic histamine concentration was measured by ELISA. Infusion of Liposyn II (4.43 kcal/3 ml) resulted in a ~3.5-fold increase in lymphatic DAO protein secretion and DAO activity, peaking at 1 h and lasting for 3 h. Liposyn II infusion also increased the lymphatic histamine release, a substrate for DAO. To determine the relationship of DAO release with histamine release, histamine was administered intraperitoneally (10 mg/kg) in fasting rats and resulted in a significant doubling in lymphatic DAO activity, supporting a link between histamine and DAO. In addition, ip administration of the histamine H4 receptor antagonist JNJ7777120 significantly reduced the Liposyn II-induced DAO output by 65.9%, whereas H(1) (pyrilamine maleate), H(2) (ranitidine), and H(3) (thioperamide maleate) receptor antagonists had little effect. We conclude that DAO secretion may contribute to the catabolism of histamine released during fat absorption and this is probably mediated through the histamine H(4) receptor.

Expression of CCL21 and 5'-Nase on pancreatic lymphatics in nonobese diabetic mice.

OBJECTIVES: To obtain better insight into the modulation of lymphatic endothelial cells during the autoimmune process, alterations of the structures and histochemical features in the pancreatic lymphatics were studied in nonobese diabetic (NOD) mice. METHODS: The expression of secondary lymphoid tissue chemokine (SLC/CCL21) and 5'-nucleotidase (5'-Nase) on pancreatic lymphatics was examined by histochemistry and immunoblot in NOD mice. RESULTS: As insulitis developed, the increased expression of CCL21 and podoplanin on pancreatic lymphatics was consistent with the increased number of cytoplasmic protrusions and vesicles, whereas 5'-Nase activity of lymphatics seemed to become decreased. The expression of CCL21 protein also showed an age-dependent increase in NOD pancreas, even though it was undetectable in normal controls. During the period of severe infiltration, reaction products of CCL21 and podoplanin were detected in the nucleus and cytoplasm of lymphatic endothelial cells. Dendritic cells and T lymphocytes frequently penetrated through the slender walls of lymphatics and adhered to the lymphatic luminal surfaces, precipitating with few 5'-Nase particles. In contrast to wild-type NOD mice, complete Freund adjuvant administration reduced CCL21 expression in NOD pancreas, suppressing the entry of activated dendritic cells into lymphatics. CONCLUSIONS: These findings suggest that CCL21 and 5'-Nase may be involved in the interaction between infiltrating cells and lymphatic vessels to induce the functional changes of lymphatic endothelial cells during insulitic and diabetic development.

Current topics of physiology and pharmacology in the lymphatic system.

We have reviewed physiological significance of rhythmical spontaneous contractions of collecting lymph vessels, which play a pivotal role in lymph transport and seem to control lymph formation through changing the pacemaker sites of the rhythmic contractions and contractile patterns of the lymphangions. A characteristic feature that the rhythmic pump activity works in vivo physiologically under the specific environment of lower oxygen tension in lymph (25-40 mm Hg) has been evaluated. With the characteristic feature, generation of endogenous nitric oxide (NO) from lymphatic endothelial cells and/or activation of ATP-sensitive potassium channels (K(ATP)) are reviewed to play crucial roles in the regulation of lymph transport at physiological or pathophysiological conditions. Chemical substances released from malignant tumor cells and tumor-derived parathyroid hormone-related peptide (PTHr-P) are also shown to cause a significant reduction of lymphatic pump activity through generation of endogenous NO and activation of K(ATP) channels. Finally, we have discussed physiological significance and roles of the lower oxygen tension in lymph, generation of endogenous NO, and activation of K(ATP) in lymph formation, lymph transport, and the functions of lymph nodes.

Endothelial nitric oxide synthase regulates microlymphatic flow via collecting lymphatics.

Functional interactions between the initial and collecting lymphatics, as well as the molecular players involved, remain elusive. In this study, we assessed the influence of nitric oxide (NO) on lymphatic fluid velocity and flow, using a mouse tail model that permits intravital microscopy and microlymphangiography. We found that NO synthase (NOS) inhibition decreased lymphatic fluid velocity in the initial lymphatics, without any effect on their morphology. Using the same model, we found a similar effect in eNOS-/- mice and in mice treated with a selective endothelial NOS (eNOS) inhibitor. Next, we uncoupled the superficial initial lymphatics from the deeper collecting lymphatics by ligating the latter and found that lymphatic fluid velocity in NOS-inhibited mice became equal to that in control animals. Surprisingly, lymphatic fluid velocity was significantly increased after ligating the collecting lymphatics, and there was a concomitant increase in injection rate and mean lymphatic vessel diameter. Our results provide the first in vivo evidence that eNOS affects function of the whole microlymphatic system and that it is regulated via the collecting lymphatics.

Expression of tissue transglutaminase in blood and lymphatic vessel endothelia and in mesothelium.

This study was undertaken to evaluate tissue transglutaminase (TG2) expression in guinea pig tissues, using an antibody against the guinea pig liver TG2. The tests were performed by means of a few immunohistochemical methods in specimens from: gut, skin, the lungs, lymph nodes, the heart, the thymus, the spleen, skeletal muscles of control guinea pigs and from inflamed skeletal muscles and skin. TG2 expression was found in artery, vein and lymphatic vessel endothelia, in mesothelia of pleura, pericardium and peritoneum, and in smooth muscle cells. Spleen deserves a special attention, since this organ, especially rich in TG2, may be much more involved in celiac disease and in liver diseases than it has been accepted so far. The subject calls for further elucidation.

Activities and expression pattern of the carbohydrate sulfotransferase GlcNAc6ST-3 (I-GlcNAc6ST): functional implications.

In recent years, a family of five GlcNAc-6-O-sulfotransferases, called the GlcNAc6STs, has been molecularly cloned. One of these, GlcNAc6ST-2 (originally named HEC-GlcNAc6ST or LSST), shows a very restricted expression at the mRNA level in high endothelial cells (HECs) of lymph nodes high endothelial venules (HEVs). This enzyme has been shown to be involved in elaborating the 6-sulfo sLex structure on a set of mucin-like acceptors within HECs, thus providing a critical recognition determinant for L-selectin during the process of lymphocyte homing to lymph nodes. Limited information has been available about the closely related sulfotransferase known as GlcNAc6ST-3 (I-GlcNAc6ST). Here, employing transfection experiments with a series of glycoprotein acceptors, we report that this sulfotransferase has a marked preference for sulfating O-linked sugars of mucin-type acceptors, whereas other sulfotransferases in the family (GlcNAc6ST-1, GlcNAc6ST-2) and a Gal-6-O-sulfotransferase exhibit strong activity on both mucin-type acceptors and glycoproteins with predominantly N-linked chains. PCR analysis of cDNAs derived from a panel of tissues and purified cell populations confirms the strong expression of GlcNAc6ST-3 in gut-associated tissues and extends the expression to include lymphocytes. In contrast to GlcNAc6ST-2, GlcNAc6ST-3 transcripts are present minimally, if at all, in HECs; moreover, this enzyme is not able to generate the 6-sulfo sLex epitope in transfected cells. These latter findings argue that GlcNAc6ST-3 is not involved in generating HEV-expressed ligands for L-selectin.

Expression of 5'-nucleotidase and alkaline phosphatase in human aqueous drainage channels.

PURPOSE: Lymphatic vessels and blood vessels can be distinguished histochemically by their expression of 5'-nucleotidase and alkaline phosphatase. The aim of this study was to compare the expression of these enzymes in human aqueous drainage channels with that seen in lymphatics and blood vessels. METHODS: Histological sections from the angular regions of human eyes were prepared both by enzyme histochemical and immunohistochemical methods to analyse 5'-nucleotidase and alkaline phosphatase expression. In some of these eyes, Indian ink-stained gelatin was injected into Schlemm's canal and the suprachoroidal space to facilitate the identification of aqueous drainage routes. RESULTS: There was no expression of 5'-nucleotidase in the endothelium of aqueous drainage channels. Ocular blood vessels were characterized by strong expression of alkaline phosphatase, whereas the cellular lining of Schlemm's canal, the collector channels, the aqueous veins and a scleral channel from the suprachoroidal space showed significantly weaker expression or no expression at all. CONCLUSION: The study failed to show a histochemical similarity between lymphatics and human aqueous drainage channels, as no expression of 5'-nucleotidase could be found in any part of the aqueous outflow pathway. The endothelium of aqueous drainage channels also differed from normal blood vessels by a much weaker expression of alkaline phosphatase. This makes a histochemical distinction between aqueous veins and scleral veins possible.

Endothelin-1 and endothelial nitric oxide synthase immunoreactivity in lymphatic vessels of the uterine broad ligament during the estrous cycle in the pig.

Immunohistochemical localization and distribution of endothelin (ET-1) and nitric oxide synthase (eNOS) were investigated in precollector and collector lymphangions of lymphatic vessels leaving the ovary and were found in the vascular subovarian plexus (mesovarium) as well as in those emanating from the oviductal isthmus and uterine horn (mesosalpinx and mesometrium, respectively) forming the paraovarian lymphatic plexus in the broad ligament of the uterus during different phases of the estrous cycle in pigs. The polyclonal antibody for ET-1 and the monoclonal antibody for eNOS isoform were used for studies on the light-microscopic level. Immunoreactivities to both ET-1 and eNOS were observed in the endothelial cell cytoplasm of precollector and collector lymphangions and were not demonstrated in smooth muscle cells of the lymphatics examined. In the endothelium, the intensity of immunostaining for ET-1 and eNOS was found to be estrous phase-dependent and differed between precollector and collector lymphangions. In general, immunoreactivity to ET-1 was more intense in the endothelium of shrunken lymphangions, whereas that for eNOS was more intense in lymphangions with the large lumen. These results suggest that ET-1 and eNOS can play a role in mechanisms regulating the vascular contractile activity promoting lymph flow during the estrous cycle in the porcine broad ligament.

[Distribution of 5'-nucleotidase activity in greater omentum of rats].

The aim of the present study is to demonstrate the cellular basis of 5'-nucleotidase (5'-Nase) activity in the greater omentum of rats. Enzyme histochemistry for 5'-Nase showed that lymphatic vessels in the omentum as well as lymphocytes in the milky spots were positively stained. Electron microscopic observation revealed-5'-Nase activity at the luminal surface of the lymphatic endothelial cells, pinocytotic vesicles in the endothelial cells and the surface of fibroblasts located at the intercellular space of adipose cells. Fibroblasts extended long cytoplasmic processes toward adipose cells and inflammatory cells. These findings suggest that lymphatic endothelial cells as well as fibroblasts in the omentum may play an important role in regulation of metabolism and immune mechanisms in the greater omentum by supplying adenosin.

Sulfation of L-selectin ligands by an HEV-restricted sulfotransferase regulates lymphocyte homing to lymph nodes.

Lymphocytes home to lymph nodes, using L-selectin to bind specific ligands on high endothelial venules (HEV). In vitro studies implicate GlcNAc-6-sulfate as an essential posttranslational modification for ligand activity. Here, we show that genetic deletion of HEC-GlcNAc6ST, a sulfotransferase that is highly restricted to HEV, results in the loss of the binding of recombinant L-selectin to the luminal aspect of HEV, elimination of lymphocyte binding in vitro, and markedly reduced in vivo homing. Reactivity with MECA 79, an adhesion-blocking mAb that stains HEV in lymph nodes and vessels in chronic inflammatory sites, is also lost from the luminal aspects of HEV. These results establish a critical role for HEC-GlcNAc6ST in lymphocyte trafficking and suggest it as an important therapeutic target.

Interleukin 1beta and GABA-transaminase activity in rat thymus.

The occurrence and distribution of 4-aminobutyrate:2 oxoglutarate transaminase (GABA-t) activity were examined in the rat thymus of normal and immunostimulated rats using biochemical and histoenzymatical methods. Specific GABA-t reactivity was confined primarily to the arteries and, to a lesser extent, to the veins. Only a few activities could be observed in association with the subcapsular and medullar part of the parenchyma and nerve fibers. GABA-t was considered a linking enzyme between the immune and the nervous system and it was studied with the aim of analyzing the relationships between these two systems. Our findings indicate that the GABA-t activity in the thymus is specifically located in the wall of the blood vessels. Moreover, our results demonstrate the presence of a GABA-t activity in the peripheral blood vessels. Treatment with interleukin 1beta induces an increase of protein content of the amounts of GABA-t biochemically assayed and of the levels of histoenzymatically stained GABA-t. Furthermore, staining of the different structures of the thymus in treated or untreated rats shows that the significant modifications concern the parenchyma, the structures resembling nerve fibers and finally, the whole thymus. On the contrary, the highest activity of the GABA-t is located in the walls of arteries, veins and lymphatic vessels.

Identification of human orbital lymphatics.

PURPOSE: To identify lymphatic vessels in the human orbit. METHODS: Lymphatic and blood capillaries were distinguished histochemically by light microscopy using a 5'-nucleotidase (5'-Nase) and alkaline phosphatase (ALPase) double staining method. Identification of lymphatic vessels was based on strict morphologic criteria combined with specific 5'-Nase staining. RESULTS: The presence of conjunctival lymphatics was confirmed and used as a control tissue. Lymphatic vessels were identified in the lacrimal gland and in the dura mater of the optic nerve. Structures demonstrating positive 5'-Nase staining at the orbital apex were highly suggestive of lymphatics but did not meet the morphologic criteria established. Lymphatic vessels were not identified in the extraocular muscles or orbital fat. CONCLUSION: To the authors' knowledge, this study presents the first evidence for lymphatic capillaries in the dura mater of the human optic nerve and lacrimal gland.

Morphometric analysis of lymphatic and blood vessels in human chronic viral liver diseases.

The purpose of this study was to elucidate the morphometric changes occurring in hepatic lymphatics in human chronic viral liver diseases and to investigate the relationship between liver fibrosis, liver inflammation, and these changes. The lymphatic vessels were stained intensely by enzyme histochemistry for 5'-nucleotidase, whereas blood vessels stained well for alkaline phosphatase. We performed a morphometric analysis to estimate the number of lymphatic and blood vessels and their areas, using computer graphics software (NIH Image). Both the number of lymphatics in the specimens and their areas were increased according to the degree of liver fibrosis, but neither showed any relationship with the degree of activity of hepatitis. Neither the number nor the areas of the blood vessels showed any obvious relationship with the degree of fibrosis or the activity of chronic hepatitis. Correlation between clinical and laboratory data and the sizes and number of the lymphatics supported these morphological data. Our results clarified that the sizes and number of lymphatics are related to the stage of fibrosis in chronic viral liver diseases. This is thought to be due to increased lymph production, which is caused by the disturbance of the microcirculation associated with liver fibrosis.

Structural organization of lymphatics in the monkey esophagus as revealed by enzyme-histochemistry.

The fine structure and distribution of lymphatic vessels in the monkey esophageal wall were studied by light and electron microscopy using an enzyme-histochemical method. Identification of lymphatics was made by 5'-nucleotidase staining, whereas blood vessels were visualized by alkaline phosphatase staining. This technique revealed intramural lymphatic networks in the mucosa, submucosa, and myenteric layer of the esophagus. The organization of lymphatics in the esophagus basically conformed to that of the small intestine, although they showed certain distribution patterns peculiar to the esophagus. The lamina propria mucosae exhibited a double-layered lymphatic network, and lymphatic capillaries extended into its papillae. Despite their forming blind ends and being closed by endothelial cells, the lymphatics in the mucosal papillae were found to contain lymphocytes in their lumen. This suggests that free cells might penetrate the endothelium to enter these initial portions of the lymphatics.

Lymphatics of the rat mammary gland during virgin, pregnant, lactating and post-weaning periods.

The distribution and ultrastructure of the lymphatics of the rat mammary gland in virgin, pregnant, lactating and post-weaning periods were examined by enzyme-histochemistry for 5'-nucleotidase (5'-Nase) and transmission electron microscopy. Enzyme-histochemistry for 5'-Nase stained lymphatics in dark brown. In the lactating period, lymphatics abounded in the interlobular connective tissues, but in other periods they were few. The interlobular lymphatics drained into collecting lymphatics running along the mammary ducts. Gaps between lymphatic endothelial cells were significantly wider in lactating period than in other periods, while both number and area of vesicles in the lymphatic endothelial cells were significantly larger in the virgin period than in other periods. In the pregnant and lactating period, the lymphatics contained many lymphocytes and lipid droplets. The results show that during lactating period, the interlobular lymphatics increase and that gaps between lymphatic endothelial cells serve as a major route through which tissue fluids and particulate matters enter the lymphatics, while vesicles seem to be main trans-endothelial transport route during the virgin period. The results will provide basic information for our next investigation on lymphangiogenesis in association with breast cancer.

Enzyme-histochemical study on postnatal development of rat stomach lymphatic vessels.

Postnatal development of rat gastric lymphatics was studied by an enzyme-histochemical method to elucidate the morphological changes of lymphatics and their relationship to maturation and function, especially in the glandular portion. The significant features of 5'-Nase-positive lymphatics in distribution and structure were examined in different stages (within 24 hr, 4-21 days, and 2 months). Lymphatics in the greater curvature and anterior wall grew much slower than those in the lesser curvature and posterior wall of the stomach in newborn and infant rats. Lymphatic islands isolated from the primary lymphatic networks in the submucosa and subserosa underwent a morphological change during this early period. This is considered one of the basic steps in lymphatic development. Occurrence of lymphatic networks in the deep lamina propria indicates that development in the gastric wall is well characterized from Day 10. With further growth and modification of lymphatics, the networks in the different layers formed an extensive communication network and many lymphatic valves were found in the submucosa and subserosa. Pinocytotic vesicles, open junctions, and intraendothelial channels were frequently detected in the mucosal and submucosal lymphatic networks of the corpus-antrum and antrum-duodenum divisional zones in the adult rats. These findings suggest that developing lymphatics in the rat stomach may represent rapidly growing tissue not only with high 5'-Nase activity but also with high adaptability for future physiological demands.