Basement membrane protein distribution in LYVE-1-immunoreactive lymphatic vessels of normal tissues and ovarian carcinomas

The endothelial cells of blood vessels assemble basement membranes that play a role in vessel formation, maintenance and function, and in the migration of inflammatory cells. However, little is known about the distribution of basement membrane constituents in lymphatic vessels. We studied the distribution of basement membrane proteins in lymphatic vessels of normal human skin, digestive tract, ovary and, as an example of tumours with abundant lymphatics, ovarian carcinomas. Basement membrane proteins were localized by immunohistochemistry with monoclonal antibodies, whereas lymphatic capillaries were detected with antibodies to the lymphatic vessel endothelial hyaluronan receptor-1, LYVE-1. In skin and ovary, fibrillar immunoreactivity for the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1 was found in the basement membrane region of the lymphatic endothelium, whereas also heterogeneous reactivity for the laminin α5 chain was detected in the digestive tract. Among ovarian carcinomas, intratumoural lymphatic vessels were found especially in endometrioid carcinomas. In addition to the laminin α4, β1, β2 and γ1 chains, type IV and XVIII collagens and nidogen-1, carcinoma lymphatics showed immunoreactivity for the laminin α5 chain and Lutheran glycoprotein, a receptor for the laminin α5 chain. In normal lymphatic capillaries, the presence of primarily α4 chain laminins may therefore compromise the formation of endothelial basement membrane, as these truncated laminins lack one of the three arms required for efficient network assembly. The localization of basement membrane proteins adjacent to lymphatic endothelia suggests a role for these proteins in lymphatic vessels. The distribution of the laminin α5 chain and Lutheran glycoprotein proposes a difference between normal and carcinoma lymphatic capillaries.     

Expression of lymphatic endothelium-specific hyaluronan receptor LYVE-1 in the developing mouse kidney

Our knowledge of the embryonic development of the lymphatic vessels within the kidney is limited. The aim of this study was to establish the time of appearance and the distribution of intra-renal lymphatic vessels in the developing mouse kidney by using the lymphatic marker, LYVE-1. Kidneys from embryonic day 12 (E12) to E18, from neonates at post-natal day 1 (P1) to P21, and from adults were studied. In the adult mouse kidney, LYVE-1 was expressed mainly in the lymphatic endothelial cells (LECs) and in a subset of endothelial cells in the glomerular capillaries. However, in the developing mouse kidney, LYVE-1 was also expressed transiently in F4/80⁺/CD11b^– immature macrophages/dendritic cells and in the developing renal vein. LYVE-1⁺ lymphatic vessels connected with extra-renal lymphatics were detected in the kidney at E13. F4/80⁺/CD11b^–/LYVE-1⁺ immature macrophages/dendritic cells appeared prior to the appearance of LYVE-1⁺ renal lymphatic vessels and were closely intermingled or even formed part of the lymphatic vascular wall. Prox1 was expressed only in the LYVE-1⁺ LECs from fetus to adult-hood, but not in LYVE-1⁺ endothelial cells of the developing renal vein and macrophages/dendritic cells. Thus, lymphatic vessels of the kidney might originate by extension of extra-renal lymphatics through an active branching process possibly associated with F4/80⁺/CD11b^–/LYVE-1⁺ macrophages/dendritic cells.     

Localisation of lymphatic vessels and vascular endothelial growth factors-C and -D in human and mouse skeletal muscle with immunohistochemistry

The present study was aimed to localise lymphatic vessels and their growth factors in human and mouse skeletal muscle with immunohistochemistry and specific antibodies (VEGFR-3, LYVE-1, VEGF-C and VEGF-D). The largest lymphatic vessels were found in perimysial connective tissue next to the arteries and veins, as has been shown earlier with electron microscopy. As a new finding, we also found small LYVE-1 positive vessels in the capillary bed between muscle fibres. These vessels were located next to CD31 positive blood capillaries and were of the same size, but fewer in number. In addition, we described the localisation of the two main lymphangiogenic growth factor proteins, vascular endothelial growth factor-C and -D. Both proteins were expressed in skeletal muscle at mRNA and protein levels. VEGF-D was located under the sarcolemma in some of the muscle fibres, in the endothelia of larger blood vessels and in fibroblasts. VEGF-C protein was localised to the nerves and muscle spindles, to fibroblasts and surrounding connective tissue, but was not found in muscle fibres or endothelial cells. Our results are the first to suggest the presence of lymphatic capillaries throughout the skeletal muscle, and to present the localisation of VEGF-C and -D in the muscles. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Enzyme-histochemical identification of lymphatic vessels by light and backscattered image scanning electron microscopy

The walls of lymphatics are characterized by strong 5'-nucleotidase activity, whereas those of blood capillaries reveal significantly lower or no activity. Alkaline phosphatase activity, on the other hand, is markedly higher in blood capillaries than in lymphatic vessels. On the basis of such characteristics, lymphatics and blood capillaries were distinguished histochemically in rat stomach using 5'-nucleotidase-alkaline phosphatase double staining. The distribution and intensity of lead-demonstrated 5'-nucleotidase activity in lymphatic vessels could be determined by comparing the images of the same histochemically stained cryostat section as seen by light and backscattered image scanning electron microscopy. The specificity of the 5'-nucleotidase reaction was obtained by inhibiting nonspecific alkaline phosphatase by including L-tetramisole in the 5'-nucleotidase incubation medium. The products of the 5'-nucleotidase activity were deposited on the outer surface of the plasma membrane of the lymphatic endothelial cells.     

Enzyme-Histochemical Identification of Lymphatic Vessels by Light and Backscattered Image Scanning Electron Microscopy

The walls of lymphatics are characterized by strong 5'-nucleotidase activity, whereas those of blood capillaries reveal significantly lower or no activity. Alkaline phosphatase activity, on the other hand, is markedly higher in blood capillaries than in lymphatic vessels. On the basis of such characteristics, lymphatics and blood capillaries were distinguished histochemically in rat stomach using 5'-nucleotidase-alkaline phosphatase double staining. The distribution and intensity of lead-demonstrated 5'-nucleotidase activity in lymphatic vessels could be determined by comparing the images of the same histochemically stained cryostat section as seen by light and backscattered image scanning electron microscopy. The specificity of the 5'-nucleotidase reaction was obtained by inhibiting nonspecific alkaline phosphatase by including L-tetramisole in the 5'-nucleotidase incubation medium. The products of the 5'-nucleotidase activity were deposited on the outer surface of the plasma membrane of the lymphatic endothelial cells.     

Lymphatic vessels in lungfishes (Dipnoi)

 Lymphatic capillaries are distributed throughout the body of lepidosirenid and protopterid Dipnoi, except in the central nervous system. They form small, interconnected units which are individually evacuated into nearby blood capillaries by lymphatic micropumps. The number of lymphatic micropumps varies considerably in different parts of the body. In fin areas, 30–50 per mm³ tissue may be considered normal in Protopterus annectens, but up to 105 per mm³ have been counted in an anterior fin of Lepidosiren paradoxa. Lymphatic capillaries are formed by thin endothelial cells with fine processes into the surrounding interstitial space. Occasionally there is a faint, discontinuous basal lamina. Pericytes, however, are completely absent. Microfibrils establish contact between endothelial cells and surrounding connective tissue fibers. The lymphatic micropumps are essentially spherical, contractile organs of 35–55 μm in diameter. Their central lumen is lined by extensions of a single endothelial cell. Additional endothelial cells form inflow and outflow valves. The endothelial layer is surrounded by a single large, highly specialized muscle cell. This spherical muscle cell has many perforations, allowing the passage of thin outward processes of the endothelial cell which form part of the suspension apparatus of the lymphatic micropump. The muscle cell establishes a specialized end-to-end contact between opposing parts of its own cell membrane. This contact is very similar to an intercalated disc in vertebrate heart muscle. Each lymphatic micropump is suspended within a cell-free tissue area by microfibrils which radiate from the lymphatic micropump into the surrounding connective tissue. The microfibrils are occasionally reinforced by single collagen fibers. The cell-free area around each lymphatic micropump appears as a bright halo in both light and electron micrographs. No type of lymphatic vessel other than lymphatic capillaries could be detected in the Dipnoi studied. Lepidosireniform Dipnoi are the only Vertebrata besides the Tetrapoda in which lymphatic vessels and characteristic lymphatic pumps have been documented. In addition, these Dipnoi and all Tetrapoda share the same overall design of blood circulation, which is not divided into a primary and a secondary system of vessels, as it is in Actinopterygii, Chondrichthyes, and Agnatha. Since there are primary and secondary blood vessels in the gills of Latimeria chalumnae, while the existence of lymphatic vessels has not been confirmed, general angioarchitecture should be taken into account as an important character when phylogenetic relationships among extant Sarcopterygii are discussed. Accepted: 7 October 1997     

Active interaction of human A375 melanoma cells with the lymphatics in vivo

We have used the avian chorioallantoic membrane (CAM) to study the interaction of tumor cells with the lymphatics in vivo. The vascular endothelial growth factor-C (VEGF-C) has been shown to be lymphangiogenic. We have therefore grown VEGF-C-expressing human A375 melanoma cells on the CAM. These tumors induced numerous lymphatics at the invasive front, and compressed or destroyed VEGF receptor (R)-3-positive lymphatics were observed within the solid tumors. The lymphatics in the CAM and in the A375 melanomas could also be demonstrated with an antibody against Prox 1, a highly specific marker of lymphatic endothelial cells. Proliferation studies revealed a BrdU labeling index of 11.6% of the lymphatic endothelial cells in the tumors and at their margins. A great number of melanoma cells invaded the lymphatics. Such interactions were not observed with VEGF-C-negative Malme 3 M melanoma cells. Lymphangiogenesis was inhibited to some extent when A375 melanoma cells were transfected with cDNA encoding soluble VEGFR-3 (sflt4), and the BrdU labeling index of the lymphatics in these tumors was 3.9%. Invasion of lymphatics and growth of blood vascular capillaries were not inhibited by the transfection. Therefore, tumor-induced lymphangiogenesis seems to be dependent to some extent on VEGF-C/flt4 interactions, but invasion of lymphatics seems to be a distinct mechanism.     

Microscopic and ultrastructural study of the lymphatic system in the human parotid gland

The localization and fine structure of the lymphatic system vessels are examined in the human parotid gland. A network of lymphatic capillaries extends in the intralobular connective septa around the striated ducts. These lymphatics converge in collectors frequently bordering the excretory ducts. On the contrary, no lymphatics are present next to the intercalated ducts and adhenomers. Ultrastructurally, the lymphatic capillaries are characterized by a very thin endothelial wall and by slightly complicated intercellular adhesions. Open junctions are also present. The presence of numerous lymphatic capillaries bordering the striated ducts and their blood microvasculature is discussed in relation to the functional activities of the striated ducts in the modification of the saliva.     

Intralobular lymphatic vessels and their relationship to blood vessels in the mouse thymus

Summary The spatial distribution and fine structure of the lymphatic vessels within the thymic lobules of normal and hydrocortisone-injected mice were studied by light- and electron microscopy. The lymphatic vessels of the cortex and medulla of normal thymus are irregularly shaped spaces closely associated with branches of the intralobular artery and vein. The overall distribution of these vessels in the greatly involuted thymus of hydrocortisone-treated mice is essentially the same as in the normal thymus. The wall of the lymphatic vessels consists of only a layer of endothelial cells supported by underlying reticular cells. The luminal surface of the endothelial cell is smooth, but trabecular processes are often seen. There are three morphological types of intercellular contacts between contiguous cells, namely, end-to-end, overlapping and interdigitating. The lymphatic vessel has anchoring filaments and collagen fibrils, but a basal lamina is either absent, or if present, is discontinuous. This is in contrast to the continuous basal lamina of the venule. The perivascular space surrounding the postcapillary venule opens into a terminal lymphatic vessel at the cortico-medullary junction and in the medulla. Lymphocytes are seen penetrating the lymphatic endothelium, particularly in acutely involuted thymuses. These findings suggest that the intralobular lymphatic vessels may originate from the vacuities that surround the postcapillary venules, and the lymphatic system may function as a pathway for the migration of lymphocytes into or out of the lymphatic circulation.     

Use of a whole-slide imaging system to assess the presence and alteration of lymphatic vessels in joint sections of arthritic mice

Abstract

We investigated the presence and alteration of lymphatic vessels in joints of arthritic mice using a whole-slide imaging system. Joints and long bone sections were cut from paraffin blocks of two mouse models of arthritis: meniscal-ligamentous injury (MLI)-induced osteoarthritis (OA) and TNF transgene (TNF-Tg)-induced rheumatoid arthritis (RA). MLI-OA mice were fed a high fat diet to accelerate OA development. TNF-Tg mice were treated with lymphatic growth factor VEGF-C virus to stimulate lymphangiogenesis. Sections were double immunofluorescence stained with anti-podoplanin and alpha-smooth muscle actin antibodies. The area and number of lymphatic capillaries and mature lymphatic vessels were determined using a whole-slide imaging system and its associated software. Lymphatic vessels in joints were distributed in soft tissues mainly around the joint capsule, ligaments, fat pads and muscles. In long bones, enriched lymphatic vessels were present in the periosteal areas adjacent to the blood vessels. Occasionally, lymphatic vessels were observed in the cortical bone. Increased lymphatic capillaries, but decreased mature lymphatic vessels, were detected in both OA and RA joints. VEGF-C treatment increased lymphatic capillary and mature vessel formation in RA joints. Our findings suggest that the lymphatic system may play an important role in arthritis pathogenesis and treatment.     

Lymphatic and Blood Vessels of the Aged Human Gingiva1

The lymphatic pathways in the gingivae from aged humans were traced by the use of the PAS reaction or iron hematoxylin stain, and their structural characteristics were compared to those of the blood microvasculature. In the young and aging gingivae the lymphatic capillaries originated in the connective tissue papillae of the lamina propria, and appeared as thin walled irregular shaped vessels. The adjacent blood capillaries in aged gingivae differed in that their walls were thicker and stained intensely Schiff positive than seen in young adult gingivae. The lymphatic capillaries emptied into thin walled collecting vessels of varying calibers that course through the lamina propria to reach the main conducting vessels that contained valves projecting within its lumen. The accompanying blood vessels were easily differentiated from the lymphatic vessels by the intense positive staining of their walls following exposure to the PAS reaction. Distended lymphatic vessels of different caliber were demonstrable in inflamed aging gingivae, suggesting that lymphatic vessels in the aged gingivae were able to provide a drainage system for excessive fluid, proteins, and other particulates from both non-injured and injured sites.     

Lymphatic vessels and Kurloff cells in the thymus of estradiol-treated guinea pigs

Summary Male guinea pigs were given a single subcutaneous injection of estradiol, which induces formation of Kurloff cells, and serial sections of thymus were examined after 10, 12, 15 and 21 days. Kurloff cells were found in large numbers in lymphatic vessels, both outside the thymus, in the interlobular tissue, at the cortical surface and inside the cortex, suggesting migration via such structures. Large extrathymic or interlobular lymphatics communicated with a previously undescribed thymic structure-the lymphatic centre-surrounded by a marginal sinus. The orientation of lymphatic valves, and the concentration of Kurloff cells within this lymphatic centre at an early time after the administration of estradiol, indicate the existence of an afferent migratory pathway. The different morphology at different times after estradiol suggest that the treatment caused a dynamic remodeling of thymic lymphatic structures.     

A scanning electron-microscopic study of the rat thymus with special reference to cell types and migration of lymphocytes into the general circulation

Summary The three-dimensional structure of the rat thymus was studied by combined scanning and transmission electron microscopy. The thymus consists mainly of four types of cells: epithelial cells, lymphocytes, macrophages, and interdigitating cells (IDCs).The epithelial cells form a meshwork in the thymus parenchyma. Cortical epithelial cells are stellate in shape, while the medullary cells comprise two types: stellate and large vacuolated elements. A continuous single layer of epithelial cells separates the parenchyma from connective tissue formations of the capsule, septa and vessels. Surrounding the blood vessels, this epithelial sheath is continuous in the cortex, while it is partly interrupted in the medulla, suggesting that the blood-thymus barrier might function more completely in the cortex.Cortical lymphocytes are round and vary in size, whereas medullary lymphocytes are mainly small, although they vary considerably in surface morphology.Two types of large wandering cells, macrophages and IDCs, could be distinguished, as well as intermediate forms. IDCs sometimes embraced or contacted lymphocytes, suggesting their role in the differentiation of the latter cells.Perivascular channels were present around venules and some arterioles in the cortico-medullary region and in the medulla. A few lymphatic vessels were present in extended perivascular spaces.The present study suggests the possible existence of two routes of passage of lymphocytes into the general circulation. One is via the lymphatics, while the other is through the postcapillary venules into the blood circulation. Our SEM images give evidence that lymphocytes use an intracellular route, i.e., the endothelium of venules.     

Pulmonary lymphatics and edema accumulation after brief lung injury

In a past study of hyperoxia-induced lung injury, the extensive lymphatic filling could have resulted from lymphatic proliferation or simple lymphatic recruitment. This study sought to determine whether brief lung injury could produce similar changes, to show which lymphatic compartments fill with edema, and to compare their three-dimensional structure. Tracheostomized rats were ventilated at high tidal volume (12-16 ml) or low tidal volume (3-5 ml) or allowed to breathe spontaneously for 25 min. Light microscopy showed more perivascular, interlobular septal, and alveolar edema in the animals ventilated at high tidal volume (P < 0.0001). Scanning electron microscopy of lymphatic casts showed extensive filling of the perivascular lymphatics in the group ventilated at high tidal volume (P < 0.01), but lymphatic filling was greater in the nonventilated group than in the group that was ventilated at low tidal volume (P < 0.01). The three-dimensional structures of the cast interlobular and perivascular lymphatics were similar. There was little filling and no difference in pleural lymphatic casts among the three groups. More edema accumulated in the surrounding lymphatics of larger blood vessels than smaller blood vessels. Brief high-tidal-volume lung injury caused pulmonary edema similar to that caused by chronic hyperoxic lung injury, except it was largely restricted to perivascular and septal lymphatics and prelymphatic spaces.     

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

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

Laminin chains in the basement membranes of human thymus

Summary In recent studies, the α2 chain of laminin (Ln) has been suggested to be the only laminin α chain expressed in mouse and human thymus. We have now used chain-specific monoclonal antibodies and indirect immunofluorescence microscopy to study the expression of laminin chains in samples of foetal and 6-year-old human thymus. The subepithelial basement membrane of the capsule of foetal 16- to 18-week thymus presented a bright immunoreactivity for Ln α1, α3, β1, β3 and γ1 chains but not for α2 chain, suggesting the expression of laminins-1 and-5. Most cortical and medullary epithelial cells, including Hassall's corpuscles, however, lacked laminin immunoreactivity. Immunoreactivity for Ln β2 chain was only seen in basal laminae of larger blood vessels. In thymic specimens from 6-year-old children, immunoreactivity for the laminin α1, α3, β1, β3 and γ1 chains was invariably found in subepithelial basement membrane of the capsule and that for laminin α2 chain was now also distinct but more heterogeneous. Furthermore, the thymic subepithelial basement membrane of the capsule at all stages showed immunore-activity for collagen type VII, forming the anchoring fibres in epithelial basement membranes. The subcapsular thymic epithelium also showed immunoreactivity for the BP 230 antigen and β₄ integrin subunit, both components of hemidesmosomes. The present results show that the thymic subepithelial basement membrane of the capsule presents properties which are commonly seen in stratified and combined epithelia, and are compatible with suggestions of the antigenic similarity of thymic epithelial cells and keratinocytes.     

Cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen

By means of immunohistochemical methods, we have investigated the cellular distribution of prothymosin alpha and parathymosin in rat thymus and spleen, using specific antibodies raised against thymosin alpha-1 and against parathymosin. We observed prothymosin alpha immunoreactivity in lymphoid cells both in thymus and spleen. In the thymus, prothymosin alpha staining was more marked in cortex than in medulla. In the spleen, prothymosin alpha was found in lymphocytes of the periarteriolar lymphatic sheaths and was especially prominent in the germinal centers. Parathymosin immunoreactivity in the thymus was mainly localized in the medulla; positive cells were reticuloepithelial cells from the thymic reticulum and the blood barrier. Thymocytes were negative. In spleen, parathymosin was found in reticular cells arranged in a ring between the periarteriolar lymphatic sheath and the marginal zone. Our results do not support an exclusive role for these peptides as immune system hormones or cytokines.     

Lymphatic capillaries in tail fin of amphibian larva: An electron microscopic study

The ultrastructure of lymphatic capillaries in the tail fin of Rana catesbiana larvae was investigated. With the use of a colloidal marker particle (Biological Carbon) the extent that these delicate vessels ramify throughout the fin region was demonstrated. This opaque substance also serves as a marker particle for identification of lymphatics with some degree of certainty at both light and electron microscopic levels. The cytoplasm of the lymphatic endothelial cell is abruptly attenuated beyond the perinuclear region, reaching widths as thin as 300 Å. Lymphatic Anchoring filaments are present, but to a lesser degree than noted for other species studied. Other features of interest include an extensive Golgi complex and electron dense bodies that are surrounded by a smooth surfaced unit membrane. These bodies are somewhat heterogeneous in size (500 Å up to 0.5 μ in diameter) and density. Numerous exit channels are provided by the extensive supply of lymphatics throughout the tail fin region of amphibian larva thus allowing them to serve an important function during metamorphosis. It is suggested that these vessels also act as passageways through which lysed cellular and connective tissue components may be rapidly removed during the process of tail fin resorption.     

Mechanisms of lymphatic regeneration after tissue transfer

Introduction

Lymphedema is the chronic swelling of an extremity that occurs commonly after lymph node resection for cancer treatment. Recent studies have demonstrated that transfer of healthy tissues can be used as a means of bypassing damaged lymphatics and ameliorating lymphedema. The purpose of these studies was to investigate the mechanisms that regulate lymphatic regeneration after tissue transfer.

Methods

Nude mice (recipients) underwent 2-mm tail skin excisions that were either left open or repaired with full-thickness skin grafts harvested from donor transgenic mice that expressed green fluorescent protein in all tissues or from LYVE-1 knockout mice. Lymphatic regeneration, expression of VEGF-C, macrophage infiltration, and potential for skin grafting to bypass damaged lymphatics were assessed.

Results

Skin grafts healed rapidly and restored lymphatic flow. Lymphatic regeneration occurred beginning at the peripheral edges of the graft, primarily from ingrowth of new lymphatic vessels originating from the recipient mouse. In addition, donor lymphatic vessels appeared to spontaneously re-anastomose with recipient vessels. Patterns of VEGF-C expression and macrophage infiltration were temporally and spatially associated with lymphatic regeneration. When compared to mice treated with excision only, there was a 4-fold decrease in tail volumes, 2.5-fold increase in lymphatic transport by lymphoscintigraphy, 40% decrease in dermal thickness, and 54% decrease in scar index in skin-grafted animals, indicating that tissue transfer could bypass damaged lymphatics and promote rapid lymphatic regeneration.

Conclusions

Our studies suggest that lymphatic regeneration after tissue transfer occurs by ingrowth of lymphatic vessels and spontaneous re-connection of existing lymphatics. This process is temporally and spatially associated with VEGF-C expression and macrophage infiltration. Finally, tissue transfer can be used to bypass damaged lymphatics and promote rapid lymphatic regeneration.     

The morphological basis of proestrus endometrial bleeding in canines

Endometrial bleeding during proestrus is a well-known phenomenon in the bitch. However, the exact events on the cellular level have not been studied. In the present investigation, immunohistochemical methods and transmission electron microscopy were employed to obtain more information about this cyclic event in canines. Long, stretched blood vessels were seen in H&E stained sections during proestrus. These vessels showed mitotic activity, as evidenced by Ki67 immunostaining. Although the endothelial lining and basement membrane of endometrial blood vessels seemed continuous, as indicated by immunohistochemical staining for laminin and Von Willebrand factor, transmission electron microscopy showed an extreme thinning and even interruption of the vascular wall in endometrial venules. Platelets were frequently seen in those areas, and also detected by immunohistochemistry. Interestingly, all endometrial capillaries examined by electron microscopy had an intact wall. We therefore postulate the endometrial venules to be the blood vessels that are mainly responsible for proestrus endometrial bleeding, rather than subepithelial capillaries.