Expression of vascular endothelial growth factor receptors in the human endometrium: modulation during the menstrual cycle

Angiogenesis is fundamental for human endometrial development and differentiation necessary for implantation. These vascular changes are thought to be mediated by the vascular endothelial growth factor (VEGF), whose specific receptors have not been examined in detail thus far. We conducted the present study to determine, by immunocytochemistry and computerized image analysis of the functionalis, the expression and modulation of the receptors Flk-1/KDR and Flt-1, which mediate VEGF effects on endothelial mitogenicity, chemotaxis, and capillary permeability. VEGF receptors are expressed mainly in endometrial endothelial cells, with variations of intensity and number of stained capillaries related to the phase of the cycle. The number of capillaries immunostained for Flk-1/KDR was maximal in the proliferative phase (ratio Flk-1/CD34: 1), twice as high as the number of Flt-1-expressing capillaries (ratio Flt-1/CD34: 0.47). The staining intensity for Flk-1 decreased during the late proliferative and early secretory phases, to increase again in the midsecretory period. The number of Flt-1-labeled capillaries was about 2-fold higher in the secretory than in the proliferative phase; however, the proportion of Flt-1-positive cells did not change, owing to the associated increase in vascular density that characterizes progression of the functionalis from the proliferative to the secretory stage. The staining intensity for Flt-1 was higher during the late proliferative and secretory phases (especially in the midsecretory phase) and the premenstrual period. In contrast, the proportion of capillaries expressing Flk-1/KDR decreased in the secretory phase (ratio Flk-1/Von Willebrand factor: 0.55). Enhanced expression of Flk-1/KDR, and of Flt-1, on narrow capillary strands at the beginning of and during the proliferative phase may account for the rapid capillary growth associated with endometrial regeneration following menstrual shedding. The high coexpression of Flk-1/KDR and Flt-1 observed on capillaries during the midsecretory period correlates with an increase of endometrial microvascular density and of permeability characteristic of this phase of the cycle, which is a prerequisite for implantation. Finally, strong expression of Flt-1, but not Flk-1/KDR, was observed on dilated capillaries during the premenstrual period and the late proliferative phase, suggesting preferential association of Flt-1 with nonproliferating capillaries at those times; activation of this receptor by VEGF could be involved in premenstrual vascular hyperpermeability, edema, and extravasation of leukocytes. In addition to the endothelial localization, we found that epithelial cells expressed Flt-1 and Flk-1/KDR. We conclude that Flt-1 and Flk-1/KDR in the functionalis are modulated in parallel or independently according to the phase of the cycle, and that these changes are responsible for VEGF actions on endometrial vascular growth and permeability. The molecular mechanisms concerning these regulations will require further investigation.     

Myocardial capillaries in the fetal and the neonatal rat: a morphometric analysis of the maturing myocardial capillary bed

Developing myocardial capillaries from 16-day-gestation fetus to adult undergo several morphological changes including a thinning of the lateral extensions of the capillary endothelial cells, the formation of a basal lamina, and an increase in the number of plasmalemmal vesicles. A decrease in the extracellular space, an increase in the number of capillaries, and a decrease in the capillary diameter were also observed during the developmental period. In view of these ultrastructural changes, a morphometric analysis was made on the developing myocardial wall to demonstrate specific quantitative changes. The volumes which were occupied by capillary endothelial cells, capillary lumina, extracellular space, and myocardial myocytes within a reference volume of myocardium were measured; and we found that 8% of the reference myocardial volume was occupied by capillary endothelial cells, 85% was occupied by myocardial myocytes, 4% was occupied by capillary lumina, and, except for a significant change in extracellular space at 16 days gestation, 3% was occupied by extracellular space. Each volume ratio was found to be nearly constant throughout the studied period. In contrast to this constancy in the volume ratios, other parameters which were measured demonstrated significant changes during the developmental period studied. These overall changes include a 135% increase in capillary density, a 63% increase in luminal surface area of capillary endothelial cells, a 24% decrease in capillary diameter, a 12% decrease in diffusion distance, and a 35% decrease in the diameter of the erythrocyte population.(ABSTRACT TRUNCATED AT 250 WORDS)     

Ultrastructural and histochemical investigation of the terminal capillaries in the spleen of the carp (Cyprinus carpio L.)

Summary In the spleen of the carp arterial capillaries of a highly differentiated structure have been studied by light and electron microscopy. These capillaries share various structural characteristics with the sheathed capillaries (ellipsoids of Schweigger-Seidel) of higher vertebrates. The long arterial capillaries of the carp spleen are provided with cuboidal endothelial cells containing filaments approximately 7 nm in diameter. There is no basal lamina. The endothelial cells form various types of cell junctions, but there are also extensive areas without any junctions. Here, a free passage is possible between the capillary lumen and the subendothelial space. The capillaries possess a single-layered sheath of macrophages. Characteristically, the sheath macrophages possess long and slender cell processes forming a loose framework, the meshes of which are filled with lymphocytes and spindle cells. The sheath macrophages show a zone of ectoplasm rich in filaments. They also contain numerous phagolysosomes rich in hydrolytic enzymes, as identified histochemically. The sheath is sharply limited against the pulp by a thick layer of collagen fibers.     

Structure of spinal cord capillaries in hypokinesia

Changes in spatial interrelations of the spinal cord capillaries and motoneurons and capillary ultrastructure were studied under hypokinesia. Spatial interrelations between the capillaries and neurons were not demonstrated to change under hypokinesia. They were estimated by the following parameters: area of neuronal profile field, number of capillaries, their length, distance from the nerve cell body, capillary bed area and index of capillary-neuronal interrelations. Quantitative investigation revealed capillary stenosis: their diameter was one and a half times less under hypokinesia. Morphologically, capillary stenosis was accompanied by the basal membrane thickening and endothelial cytoplasm vacuolization. There was a direct relation between endothelial villi and the places of the endothelial cells contacts, dilatation of the contact interstices and solidifying of their borders. Changes in the capillaries were followed by reactions in the pericapillary structures, such as: fibrillae were formed, mitochondrii accumulated in the perivascular glial projections, the membrane next to capillary astrocyte projections underwent desmosome-like condensation. Mitochondrial accumulations were also observed in the nerve cell projections and in their cytoplasm sites contacting with the blood vessels.     

Ovarian steroids in endometrial angiogenesis

Angiogenesis, the sprouting of new blood vessels from pre-existing ones, is fundamental for human endometrial development and differentiation, which are necessary for implantation. This vascular process is supposed to be mainly mediated by the vascular endothelial growth factor (VEGF), also named vascular permeability factor (VPF). We report here the expression and modulation of VEGF and its receptors, Flk-1/KDR and Flt-1, in the functionalis throughout the menstrual cycle. Using immunocytochemistry, VEGF is localized in glandular epithelial cells and in the surrounding stroma, as well as in capillaries and spiral arterioles. The localization of VEGF on the endothelium correlates with the presence of Flt-1 and Flk-1/KDR receptors on vascular structures, including capillary strands that have not yet formed a lumen and that have been previously described in tumors as angiogenic capillaries. The strongest immunoreactivity for both VEGF and Flk-1/KDR receptor on endothelial cells is detected in the proliferative and midsecretory phases. Enhanced expression of VEGF and its Flk-1 receptors on narrow capillary strands during the proliferative phase may account for the rapid capillary growth associated with endometrial regeneration from the residual basal layer following menstrual shedding of the functionalis. The vascular expression of Flt-1 is more important in the secretory than in the proliferative phase, associated with a high microvascular density and an increase in vascular permeability in the implantation period. Consistently with these in vivo observations, the treatment of isolated endometrial stromal cells with estradiol (E(2)), or E(2) + progesterone, significantly increased VEGF mRNA over the control value in a dose-dependent manner. These results demonstrate that the expression of VEGF and its receptors is cyclically modulated by ovarian steroids, and that this endothelial growth factor acts on the endothelium in a paracrine fashion to control endometrial angiogenesis and permeability.     

The role of the parenchyma sheath and PCD during the development of oil cavities in Pterodon pubescens (Leguminosae-Papilionoideae)

Pterodon pubescens cavities are constituted by lumen and uniseriated epithelium surrounded by multiseriate parenchyma sheath. We studied the development of secretory cavities, including the role of parenchyma sheath, using light and transmission electron microscopy. A Tunel assay was performed to verify whether programmed cell death (PCD) occurs during the process. The lumen is formed by schizogeny and lysigeny occur in later developmental stages of the secretory cavities. Ultrastructurally, epithelial cells in later developmental stages become dark and with sinuous walls; the protoplast becomes retracted and the cytoplasm shows low organelle definition. Degenerated cells are released toward the lumen. Our results showed that PCD occurs during later developmental stages of cavities and plays a critical role in functioning of these glands. New cells originated from the parenchyma sheath differentiate into secretory cells and replace those degenerated ones. This fact associated to PCD guarantees epithelium renovation during the secretory cycle and the maintenance of secretory activity of cavities.     

A quantitative investigation of microvascular changes in the thyroid gland after infrared (IR) laser radiation.

We present an ultrastructural study of thyroid capillaries in which 50-day-old rats Wistar rats, were irradiated with an infrared (IR) laser, (total dose, 46.80 J/cm2), the tissue quantified 1 day after ending treatment and a quantitative capillary analysis carried out by light and electron microscopy. Light microscopy was used to calculate capillary volume density revealing a significant increase in the irradiated rats. The quantitative measurement of parameters by electron microscopy required a two stage analysis: Level I, Electron Microscopy (Magnification x5,000); and Level II, Electron Microscopy (Magnification x26,000). At Level I, the following parameters were measured in each capillary: capillary area, capillary diameter, luminal area, luminal diameter, endothelial area, nuclear area and mean endothelial thickness. At Level II, pinocytotic vesicle diameter and their numerical density in endothelial cells were evaluated. Electron microscopic analysis revealed an increased luminal area in the capillaries of the irradiated rats. They also presented a decrease in endothelial cell thickness and vesicular diameter and an increase in vesicle numerical density. This latter increase is indicative of presumptive changes in capillary permeability, but the possible functional significance of these morphological changes in the endothelial cells requires further investigation.     

Changes in glomerular structure after sexual maturation and seawater adaptation in males of the euryhaline teleost Gasterosteus aculeatus L.

Summary In sexually mature male sticklebacks, the renal tubular cells are transformed from ion reabsorbing to mucus secreting cells and in these fish concomitant changes take place in the glomeruli.The present study compares glomerular structure of immature males in fresh water (controls) to those of mature males in fresh water and to immature male sticklebacks in seawater. Glomerular structure is markedly altered in the latter two groups and the changes are similar to a large extent. In these two groups the renal capsules and glomeruli are smaller and the lumina of the glomerular capillaries decrease in diameter, while the number and size of the endothelial fenestrations are reduced. Mesangial cells proliferate and the mesangial matrix greatly expands in both the centrolobular region and the subendothelial space around the capillaries. The secretory activity of the podocytes is enhanced and is responsible for the observed increase in thickness of the outer layer of the basal lamina, the lamina rara externa. The area covered by the filtration slit membranes is reduced, probably as a consequence of fusion of the pedicels of the podocytes. The permeability characteristics of the glomerular filtration barrier for macromolecules, as studied with ferritin injections, remain unaltered. However, the observed differences point to a reduction of the glomerular filtration rate (GFR) during maturation in male sticklebacks, as well as during adaptation of sticklebacks to seawater. This conclusion is in line with physiological evidence.     

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.     

Morphological changes in the oviductal endometrium during the reproductive cycle of the tortoise,Gopherus polyphemus

The oviducts of 24 tortoises (Gopherus polyphemus) were examined using histological techniques and scanning electron microscopy to determine endometrial morphology. Measurements of endometrial characteristics (epithelial cell height, cilia length, thickness of endometrial glandular layer, and glandular diameter) in the uterus and tube (tuba uterina) were obtained to determine changes during the reproductive cycle. Epithelial cell height increases in both the uterus and the tube during vitellogenesis and remains hypertrophied during gravidity. Cilia length increases in the uterus during late vitellogenesis and gravidity, but the length of tubal cilia does not change during the reproductive cycle. The ratio of secretory to ciliated epithelial cells in the oviduct increases from quiescence to gravidity. The thickness of the glandular endometrial layer increases in both the uterus and tube during vitellogenesis. In the uterus, the glandular layer decreases in thickness during gravidity. The diameter of the uterine glands increases throughout vitellogenesis and gravidity; however, following ovulation glandular cells become depleted of secretory granules and cell height diminishes. The diameter of the tubal glands is unchanged during the reproductive cycle. Oviductal hypertrophy during vitellogenesis coincides with elevated circulating estradiol, whereas during gravidity progesterone concentrations peak (Taylor, '82, PhD Dissertation, University of Florida, Gainesville) and may induce secretion of albumen and eggshell components.     

Ultrastructural changes in the oviductal epithelium of Merino ewes during the estrous cycle

Isthmic and ampullary oviductal epithelia sampled from Merino ewes at days -1, 1, 3, and 10 of the estrous cycle (estrus = day 0) were studied by scanning and transmission electron microscopy after fixation by vascular perfusion. Secretory cells, ciliated cells, and lymphocytelike basal cells were observed in both isthmic and ampullary epithelium at all stages of the estrous cycle studied and their ultrastructural features were analyzed. Synthesis of lamellated secretory granules occurred in the ampullary secretory cells during the follicular and early luteal phases, and their contents were released by exocytosis into the oviductal lumen during the luteal phase. Granule release was associated with nucleated apical protrusion of these cells into the oviductal lumen. No such secretory activity was displayed by isthmic secretory cells even though a few cells contained nonlamellated granules. Apocrine release of apical vesicles and accompanying cytoplasmic material from apical protrusions of ciliated cells occurred in the isthmus around estrus but not in the ampulla. This unexpected feature has not previously been reported in any other mammal. Dendritic basal cells were distinguished in the lower part of the epithelium by their heterochromatic nuclei, electron-lucent cytoplasm, and lack of attachment zones. No migration of basal cells was observed, and their ultrastructural features were similar in the ampulla and isthmus and at all stages of the estrous cycle examined. The function of these lymphocytelike cells in the epithelium is uncertain, but the presence of phagocytic bodies and lysosomes in 20% of them may indicate a phagocytic role.     

Immunoglobulins in the mouse uterus during the oestrous cycle

The distribution of IgA, IgG and IgM was studied by an immunoperoxidase technique on sections of mouse uteri at each stage of the oestrous cycle. Staining for IgG and IgA was highest at pro-oestrus, declined at oestrus and was very low during the other stages. At pro-oestrus IgG was found throughout the stroma, in the uterine lumen, and in 10% of glandular lumina; very few IgG-containing plasma cells were present. At pro-oestrus, IgA was found in the uterine lumen, and in most of the uterine glands, both in the lumen and in the epithelium; little IgA was present in the stroma. IgA-plasma cells were detected at each stage of the cycle and were particularly numerous at pro-oestrus and oestrus. These results suggest that IgA is secreted locally from plasma cells into the uterine gland through the glandular epithelium, but that IgG enters the stroma from the local capillaries. The obvious increase in IgG and IgA secretion at pro-oestrus, when plasma oestradiol levels are highest, supports the hypothesis that, during the oestrous cycle, the humoral immune response is regulated in the uterus by ovarian hormones.     

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     

Microvascular development: learning from pancreatic islets

Microvascular development is determined by the interplay between tissue cells and microvascular endothelial cells. Because the pancreatic islet is an organ composed mainly of endothelial and endocrine cells, it represents a good model tissue for studying microvascular development in the context of a tissue. In this review, we will describe the special morphology of islet capillaries and its role in the physiologic function of islets: secretion of insulin in response to blood glucose levels. We will speculate on how islet-secreted VEGF-A generates a permeable endothelium that allows insulin to pass quickly into the blood stream. In addition, we speculate on how endothelial cells might form a capillary lumen within the islets. At the end, we look at the islet microvasculature from a medical point of view, thus describing its critical role during type I diabetes and islet transplantation.     

Light- and electron-microscopic study on the maturation of the primary portal plexus during the perinatal period in rats

The maturation of the capillaries of the primary portal plexus in rats during the perinatal period has been studied light- and electron-microscopically. The number of capillaries covering the median eminence and of those invading the nervous tissue (capillary loops) increases significantly with age. Capillary loops were observed as early as the 18th fetal day. The mitotic divisions of the endothelial cells within the preexisting capillaries seem to be the main reason for the vascular growth. Immature capillaries with a characteristic narrow lumen are surrounded by a fuzzy basal lamina; their wall is formed by a generally expanded endothelium with rather sparse organelles and inclusions, and by minute flattened areas. The maturation of the capillary results in a progressive spread of flattened endothelium followed by an enlargment of the capillary lumen. Moreover, a rising concentration of organelles and inclusions, relatively numerous luminal microvilli, and a dense and uniform basal lamina become noticeable as capillary differentiation proceeds. These data are thought to reflect the progressive increase in the metabolic activity of the endothelium as well as the establishment of capillary patency during the perinatal period of rats.     

Observations on the morphology of pancreatic secretory capillaries

Summary The fine morphology of, and the light microscopic distribution of ATPase in, pancreatic secretory capillaries suggest that they may be absorptive channels receiving material from the general blood circulation rather than secretory channels communicating with the duct system. This assumption is based upon many observations; 1. The pattern of microvilli of their wall differs from that of acinar lumen. 2. The material retained by them in ultrathin sections differs from that retained by acinar lumen. 3. They ramify, basally, to the level of Golgi region. 4. The lack of release of zymogen droplets in them. 5. Their points of origin from acinar lumen are controlled by zonulae occludentes. 6. Gulf-like extensions from the basal surface of acinar cell communicate with them. 7. Fenestrated blood capillaries run in their vicinity. 8. Blood capillaries and acinar cells share a joint basement lamina. 9. Light microscopically, ATPase activity is associated with their wall and not with the basal acinar cell surface.Publication No. 932 of the Division of Basic Health Sciences of Emory University; which was supported by the McCandless Research Funds and the School of Dentistry Fellowship Funds, of Emory University. The technical assistance of Miss Brenda Callahan, Mr. Peter Daly and Mr. Hugo Greiner is acknowledged. The use of the facilities of Dr. Wallace G. Campbell Jr. is appreciated.     

Light and electron microscopic studies of the murine heart after repeated administrations of ciguatoxin or ciguatoxin-4c.

After repeated ip and oral administrations of ciguatoxin (CTX) and ciguatoxin-4c (CTX-4c), one of the derivatives of CTX, to male ICR mice at a dose of 0.1 microgram/kg for 15 days, resulted in marked swelling of cardiac cells and endothelial lining cells of blood capillaries in the heart was observed. Single doses caused no discernible pathological changes. Damage to the capillaries was followed by prominent effusion of serum and erythrocytes into the interstitial spaces of the myocardium occurred. Swelling of the endothelial lining cells of capillaries caused narrowing of the lumen and accumulation of blood platelets in capillaries, which resulted in multiple single cell necroses of cardiac muscle cells. Within 1 month after the treatments of these phycotoxins, myocytes and capillaries appeared to be normal. Effusion in the interstitial spaces resulted in formation of bundles of dense collagen, which persisted for 14 months. Diffuse interstitial fibrosis was prominent in septum and ventricles, accompanied by bilateral ventricular hypertrophy. A single dose of 0.7 micrograms/kg ip resulted in severe acute heart injuries, followed by diffuse myocardial fibrosis.     

Electron microscope identification of endocrine secretory granules in endothelial cells

Electron microscopy has revealed endocrine secretory granules in the endothelial cells of the rat liver, spleen and bone marrow capillaries. The granules were responsible for serotonin, melatonin, catecholamines and insulin synthesis. Local mechanisms of hormonal control of hemostasis are discussed.     

Changes in swimbladder retial morphology in Anguilla rostrata during premigration metamorphosis

During metamorphosis from the yellow to the silver phase in Anguilla rostrata , the length of the swimbladder retial capillaries increases by a factor of 2.5, the mean retial capillary lumen diameter and total retial cros-sectional area increase by 1.5 and the calculated counter current exchange efficiency increases by 3.4. Metamorphosis of the retia begins before changes in integument pigmentation become apparent. These changes in retial morphology are part of a complex of transformations that permit. The maintenance to swimbladder function at greater depths during the spawning migration.     

The marker histochemical detection of glucose-6-phosphate residues in the human placenta]

Glucose-6-phosphate residues were revealed by fine structural analysis using glucose-6-phosphate dehydrogenase-gold conjugate. In human mature placenta specific staining was detected over the stroma of placental villi. Colloidal gold particles were found over the collagen fibrils and reticular lamina of basal membrane. Syncytiotrophoblast cells, fibroblasts, endothelial cells of fetal capillaries avoided labelling. Nucleated blood cells and thrombocytes inside the lumen of fetal capillaries demonstrated intense staining. The present investigation demonstrates histochemically the process of extracellular glycosylation. Glycated collagen fibrils formed channels inside the stroma of placental villi.