Quantitative immunocytochemical studies of endogenous albumin in rat aortic endothelial and mesothelial cells

Endogenous albumin was revealed over cellular structures of rat ascendent aorta endothelia and mesothelium, with high resolution and specificity, by applying the protein A-gold immunocytochemical approach. This approach allows albumin distribution to be studied under steady-state conditions. The cellular layers evaluated were the aortic endothelium, the capillary endothelium (vasa vasorum), and the mesothelium externally lining the aorta at this level. Gold particles, revealing albumin antigenic sites, were preferentially located over plasmalemmal vesicles and intercellular clefts of endothelial and mesothelial cells, though with different labeling intensities. The interstitial space was also labeled. Morphometrical evaluation of plasmalemmal vesicles demonstrated a higher surface density for these structures in capillary endothelial cells (12%) compared with those in aortic endothelial (5%) and mesothelial cells (2%). Quantitation of gold labeling intensities over these structures revealed a higher labeling over plasmalemmal vesicles of capillary endothelium than over those of aortic endothelium and mesothelium. This result, together with the higher surface density of plasmalemmal vesicles found in capillary endothelium, suggest an important role of these structures in the transendothelial passage of endogenous albumin, particularly for capillary endothelium. On the other hand, labeling densities over mesothelial clefts were found to be higher than those of capillary and aortic endothelia. Results from this study concur with the proposal of a differential passage of albumin according to the cell lining considered, and suggest to a role for mesothelial intercellular clefts in contributing to the presence of albumin in interstitial spaces.     

Presence of α-smooth muscle actin-positive endothelial cells in the luminal surface of adult aorta

α-Smooth muscle actin-positive endothelial cells have not been found in adult aortic endothelium except valve leaflets. Here, using en face immunostaining method, we identified α-smooth muscle actin-positive endothelial cells in the luminal surface of rat, mouse and human thoracic aortas. These cells express both endothelial markers and definite smooth muscle cell markers and were only occasionally observed in thoracic aorta of wild type mice and rats. Their density did not increase with aging. Given that α-smooth muscle actin-positive endothelial cells express low level of vascular endothelial-cadherin that is important for the maintenance of cell contact, these cells were frequently detected in the thoracic aorta of 5-week-old apolipoprotein-E deficient mice. In 20- to 24-week-old apolipoprotein-E deficient mice, marked accumulation of α-smooth muscle actin-positive endothelial cells was observed especially in the luminal surface of atheromatous plaques. Our findings indicate the existence of α-smooth muscle actin-positive endothelial cells in adult aortic endothelium and the possible association with progression of atherosclerosis.     

Endothelial layers in fish skin

Layers of cells limiting the deep face of the dermis and lining the scale pockets can be described as endothelial, using the term in the broad sense. A dermal endothelium has been found in lampreys and in teleosts of diverse form and habits; it consists of a single layer of modified fibrocytes joined by desmosomal and other junctions and having hemidesmosomes and numerous caveolae intracellulares . A fibrous zone interpreted as elastic tissue intervenes between the dermal endothelium and the collagen of the stratum compactum . The scale pocket lining consists of cells with caveolae, desmosomes, hemidesmosomes and usually with basement membrane. The lining may be one or two cells thick and may occur on both aspects of the scale pocket or only on the deeper side, depending on the species. The fine structure of these endothelial layers is compared with that of the vascular and lymphatic endothelia, the scale-forming cells, the perineurium and the peritoneal lining.     

Microvascular polytetrafluoroethylene prostheses: the cellular events of healing and prostacyclin production

This study was undertaken to obtain more insight into the morphologic and functional performance of microvascular polytetrafluoroethylene (PTFE) prostheses. Therefore, both the cellular events of healing (n = 30) and the prostacyclin production (n = 18) of microvascular polytetrafluoroethylene prostheses (length 10 mm, internal diameter 1.5 mm) were evaluated from 1 hour up to 3 months after implantation into the abdominal aorta of rats. After implantation, the graft surface became scarcely covered with platelets. From 1 week onward, endothelial cells originating from the anastomotic sides grew in over the graft surface, covered only about half the prostheses after 3 months of implantation, but did produce normal amounts of prostacyclin as compared to normal endothelium. Only near the anastomotic sides one to two layers of smooth-muscle-like cells developed underneath the neoendothelial lining. Perigraft tissue ingrowth into the wall of the polytetrafluoroethylene prostheses was scarce. The overall patency rate was 98 percent. It was concluded that optimalization of the healing characteristics of microvascular polytetrafluoroethylene prostheses may provide a prosthesis that is more suitable for clinical microsurgery.     

Blood-forming potential of vascular endothelium in the human embryo

Hematopoietic cells arise first in the third week of human ontogeny inside yolk sac developing blood vessels, then, one week later and independently, from the wall of the embryonic aorta and vitelline artery. To address the suggested derivation of emerging hematopoietic stem cells from the vessel endothelium, endothelial cells have been sorted by flow cytometry from the yolk sac and aorta and cultured in the presence of stromal cells that support human multilineage hematopoiesis. Embryonic endothelial cells were most accurately selected on CD34 or CD31 surface expression and absence of CD45, which guaranteed the absence of contaminating hematopoietic cells. Yet, rigorously selected endothelial cells yielded a progeny of myelo-lymphoid cells in culture. The frequency of hemogenic endothelial cells in the yolk sac and aorta reflected the actual blood-forming activity of these tissues, as a function of developmental age. Even less expected, a subset of endothelial cells sorted similarly from the embryonic liver and fetal bone marrow also exhibited blood-forming potential. These results suggest that a part at least of emerging hematopoietic cells in the human embryo and fetus originate in vascular walls.     

In situ fluorescence labeling of sheep lung microvascular endothelium

Summary Endothelial cells are intimately involved in a variety of biological processes such as inflammatory disorders, wound healing, and tumor invasion. The finding of endothelial heterogeneity in various tissues has led to major efforts to isolate and culture microvascular endothelial cells in human and animal tissue. In this report we have used phosphatidyl ethanolamine (PE)-labeled liposomes to fluorescently label the sheep lung microvasculature in situ. Using normotensive perfusion pressure, the PE-labeled liposomes did not extravasate into extravascular lung tissue. Mechanical and enzymatic digestion of the lung tissue demonstrated that the PE-labeled liposomes provided a stable label of the vascular lining cells during ex vivo processing. After digestion, the overwhelming majority of the fluorescent label appeared in cellular aggregates. Approximately 80% of these cells demonstrated an in vitro phenotype consistent with microvascular endothelium. A novel monoclonal antibody selective for sheep endothelial cells was developed to confirm the presence of lung endothelium in the fluorescently labeled cellular aggregates. We conclude that in situ fluorescence labeling of vascular lining cells provides an anatomic marker for relevant vascular lining cells and an opportunity to study these cells in vitro.     

Endothelium of the jugular vein of domestic chicken at the remote periods after injury]

Detailed analysis of changes in the endothelial layer at late terms after injury revealed that in one month after cauterization with a 0,25%solution of silver nitrate the recovered epithelium was not substatially different from the initial state by most features and retained its specific properties. From the normal state itdistinguished by the cell size, their orientation with respect to the longitudinal axis ofthe vessel, but the signs characterizing the lining as a whole, such as correct arrangement of the layer, the shape of the cells, gave evidence that the endothelium had recovered retainingits specific properties as a highly sensitive system with intrinsic signs of a tissue type. A significant amount of binuclear cells at late terms as well as greater amount of nuclei with centromeres as compared with the normal state pointed out that the traces of the reactive processretained for a very long time.     

Incorporation of adult organ-derived endothelial cells into tumor blood vessel

In this study, we attempted to assess the incorporable potential of vascular endothelial cells derived from adult organ blood vessels into tumor blood vessels. Two kinds of adult organ-derived vascular endothelial cells, human aorta endothelial cells (HAEC) and umbilical vein endothelial cells (HUVEC), were administered into murine tumors inoculated to SCID mice. Many human blood vessel networks were visualized in the murine tumors. These cells in solid tumor not only survived and proliferated, but also incorporated into tumor endothelium. These results suggest that adult organ-derived vascular endothelial cells possess the potential to form the neovascular network in various tissues such as vascular endothelial progenitor-like cells in vivo. We propose that these cells can be regarded as a congenic (autologous) vector for vascular regeneration cell therapy and tumor vascular targeting gene therapy.     

Buthionine sulfoximine causes endothelium dependent hyper-relaxation and hypoadiponectinemia

A close relationship between oxidative stress, endothelial dysfunction, and hypoadiponectinemia has been observed. The present study was performed to investigate how glutathione depletion via buthionine sulfoximine (BSO) administration affects endothelial function and adiponectin levels in rats. Acetylcholine (Ach)-induced vasodilation was significantly enhanced in BSO-treated rats, compared with control rats. This was completely abolished by L-NAME, and Ach-induced vasodilation was not observed in the aorta without endothelium. These results suggest that Ach-induced hyper-relaxation of the aorta in BSO-treated rats is completely dependent on the presence of endothelium and mediated by changes in eNOS activity. Catalase significantly inhibited this relaxation to Ach and no effect of catalase on sodium nitroprusside-induced relaxation of the aorta without endothelium was observed in BSO-treated rats. Thus, hyper-relaxation of the aorta in BSO-treated rats is likely caused by H₂O₂ in addition to NO produced by the endothelium via an eNOS-dependent mechanism. Hypoadiponectinemia and decreased levels of adiponectin mRNA in adipose tissue were observed in BSO-treated rats. Protein expression of eNOS and SODs (SOD-1 and SOD-2) in the aorta was increased and plasma NOx levels were decreased in BSO-treated rats. Our results suggest that oxidative stress induced by BSO causes eNOS uncoupling and hyper-relaxation by producing H₂O₂, and that BSO-induced oxidative stress causes hypoadiponectinemia, probably by increasing H₂O₂ production in adipose tissue.     

Endocardial endothelium in the rat: cell shape and organization of the cytoskeleton

The cytoskeleton in endocardial endothelium of rat heart was examined by en face confocal scanning laser microscopy. In the ventricular cavity, endocardial endothelial cells had a polygonal shape and F-actin staining was generally restricted to the peripheral junctional actin band. Central F-actin bundles, or stress fibers, in endocardial endothelial cells were found on the tendon end of papillary muscles, especially in the right ventricle, and frequently in the outflow tract of both ventricles; elsewhere, stress fibers were scarce. Many endocardial endothelial cells were elongated in areas of endothelium with stress fibers, but no correlation was found between cell elongation and the number of stress fibers. An inverse correlation was found between the number of stress fibers and the surface area of endocardial endothelial cells. Shear stress as well as mechanical deformation of the surface of the ventricular wall during the cardiac cycle may affect cell shape and the organization of actin filaments in endocardial endothelial cells. Vimentin in endocardial endothelial cells formed a filamentous network with some distinct cytoplasmic and juxtanuclear vimentin bundles. No perinuclear ring of vimentin filaments was observed in endocardial endothelium. Microtubules in endocardial endothelial cells were, in contrast to endothelial cells of rat aorta, not aligned, less closely packed and originated from randomly distributed centriolar regions. The cytoskeleton has been suggested to play an important role in cellular functions of vascular endothelial cells. Accordingly, differences in the cytoskeletal organization between endocardial and vascular endothelial cells may relate to differences in functional properties.     

Protein expression profiles of Bos taurus blood and lymphatic vessel endothelial cells

The endothelium is a metabolically active organ that regulates the interaction between blood or lymph and the vessel or the surrounding tissue. Blood endothelium has been the object of many investigations whereas lymphatic endothelium biology is yet poorly understood. This report deals with a proteomic approach to the characterization and comparative analysis of lymphatic and blood vessel endothelial cells (ECs). By 2-DE we visualized the protein profiles of EC extracts from the thoracic aorta, inferior vena cava, and thoracic duct of Bos taurus. The three obtained electropherograms were then analyzed by specific software, and 113 quantitative and 25 qualitative differences were detected between the three endothelial gels. The cluster analysis of qualitative and quantitative differences evidenced the protein pattern of lymphatic ECs to be more similar to the venous than to the arterial one. Moreover, venous ECs were interestingly found showing a protein expression profile more similar to the lymphatic ECs than to the arterial ones. We also identified 64 protein spots by MALDI-TOF MS and ESI-IT MS/MS and three reference maps of bovine endothelium were obtained. The functional implications of the identified proteins in vascular endothelial biology are discussed.     

IN VIVO ORGAN RECONSTRUCTION FROM CELLS CULTURED IN VITRO

Dispersed primary tissue culture cells from adrenal, aorta, heart and cornea of adult rabbits were placed into diffusion chambers. The chambers were implanted into serologically compatible litter mates. Chimaeric aggregates resembling embryonal organs formed in the chambers within 3 weeks. The ‘adrenal’had two to three cortical zones to both sides of a medulla. The ‘aorta’had a central fibromuscular layer and a surface layer of endothelial cells. The ‘heart’consisted of a muscular layer coated with endothelial cells; the aggregate assumed a chamber-like shape. The ‘cornea’had a loose matrix covered on the convexity by epithelial cells. Upon transfer of the aggregates from in vivo to in vitro culture, cells dispersed within 1 week and propagated as a mixed population. Analysis of the phenomenon has been attempted and the course of future studies suggested. The study contributes to the discussion of cell automatism.     

Characterisation of the interaction between circulating and in vitro cultivated endothelial progenitor cells and the endothelial barrier

In vitro cultured endothelial progenitor cells (cEPC) are used for intracoronary cell therapy in cardiac regeneration. The aim of this study was to investigate whether cEPC and circulating mononuclear cells (MNC), which include a small number of in vivo circulating EPC, are able to transmigrate through the endothelial barrier into the cardiac tissue. MNC and EPC were isolated from the peripheral blood from healthy male volunteers (n = 13, 25+/-6 years) and stained with a fluorescent marker. The cells were perfused in vitro through organs with endothelial layers of different phenotypes (rat aorta, human umbilical vein, isolated mouse heart). The endothelium and the basal lamina were then stained by immunofluorescence and the cryo-sections analysed using a confocal laser scanning microscope. After perfusion through the rat aorta, an adhesion/integration of MNC was observed at the endothelial layer and the basal lamina beneath endothelial cells. However, no migration of MNC over the endothelial barrier was found. This remained true even when the cell numbers were increased (from 0.5 to 10 million cells/h), when the time of perfusion was prolonged (1.5-4 h) and when the aorta was cultivated for 24 h. In the Langendorff-perfused mouse heart with intact endothelium, no migration of MNC (1 x 10(7)) or cEPC (1 x 10(6)) was observed after 0.5 and 2 h. In conclusion, MNC and cEPC do not possess any capacity to transmigrate the endothelial barrier. In the context of stem cell therapy, these cells may therefore serve as endothelial regenerators but not as cardiomyocyte substitutes.     

The distribution of centrosomes in migrating endothelial cells during wound healing in situ.

We have examined the distribution of centrioles in rabbit thoracic aortic endothelial cells induced to migrate by wounding the endothelium in situ. Following denudation of the endothelium from a segment of the aorta with a balloon catheter, a wound edge was created from which endothelial cells began to migrate onto the denuded surface. In this in situ model of cell migration, the position of centrioles was determined in cells along the wound edge by immunofluorescence and antibodies which specifically label these cell organelles, and then they were classified in relation to the nucleus and the direction of cell migration as being oriented toward the wound, in the center, or away from wound. At time 0, as in normal unwounded adult rabbit aorta, no preferential orientation of centrioles was evident. Within 12 h after wounding, the centrioles in about 53% of endothelial cells near the wound edge were oriented toward the wound, while in less than 20% of the cells they were oriented away from wound. At 24 h, in cells up to 800 microns from the wound edge, centrioles in only about 10% of the endothelial cells were oriented away from wound, while in about 52% of cells they were found in the center and in 38% of the cells they remained oriented toward the wound. At 48 h, up to 2000 microns from the wound edge, the majority of endothelial cells had their centrioles in the center, possibly as a result of an increase in mitotic index as cells replicate to reestablish an intact endothelium.(ABSTRACT TRUNCATED AT 250 WORDS)     

Tissue nature of the lining of the lymph node sinuses]

The lymphatic nodes of intact albino rats were investigated electron microscopically. It was shown that the lymphatic sinuses were restricted by a layer of flattened cells; the basal membrane was absent. Certain distinctions in the structure of the cell lining sinuses and the reticular cells comprising the reticular base of the lymphoid tissue of the lymphatic node were found. The structure of the "sinus network" strands is shown. The structure of the cells of the sinuses lining is shown to be identical to the structure of cells of the vascular endothelium. It suggests the endothelial nature of the lining of the lymphatic node sinuses.     

Comparative histochemical and electron microscopic studies of the sinus and venous walls of the human spleen with special reference to the sinus-venous connections

Sinus and venous walls of normal human spleens were studied with enzyme histochemical and electron microscopic methods. Particular attention was paid to the connections between sinuses and veins. Histochemically the sinus lining cells revealed a distinct naphthol-AS-acetate-esterase activity but no reaction for alkaline phosphatase. Venous endothelial cells were positive for the latter but negative for the former enzyme. In the sinus-venous junctional area there were no endothelial cells with reactivity for both enzymes. Electron microscopically both the sinus lining cells and the venous endothelial cells could be clearly characterized and therefore easily distinguished from one another on morphological grounds. There were no clear ultrastrural indications of transitional forms between sinus lining cells and venous endothelial cells in the sinus-venous area. According to these findings, sinus lining cells represent a specialized endothelium, but one with practically no morphological similarities to the venous endothelium.     

Reactivity of the aorta from spontaneously hypertensive rats before and after endothelial removal

In the present study the mechanic activity of SHR and Wistar rat's aorta was evaluated, in vitro, after stimulation by chloride of potassium, phenylephrine, norepinephrine, histamine, serotonin and acetylcholine, before and after the removal of the endothelium. The aorta rings of the rats were taken before the development of the hypertensive state (7th week) and at 18th week (when the SHR rats already showed established hypertension starting since IXth week of life), and successively suspended in a bath for isolated organ. The mechanic activity was measured by isometric transductor. The obtained findings show an increased sensitivity, in SHRs, to (K+), NA and FeE, if these are compared with the control group, since the prehypertensive stage (7th week). The removal of the endothelium didn't modify the response amplitude to K in both the breeds, while the maximum response amplitude, provoked by NA and FeE, significantly increased in SHRs compared to controls. The relaxation induced by the vasodilator agents (Ach-H-5HT) was completely absent in the SHR rats' aorta with established hypertension. In conclusion, these results suggest a functional deterioration of the endothelial cells, in the hypertensive animals, that could contribute to increase the peripheric vascular resistances observed during hypertension.     

Lectin binding to injured corneal endothelium mimics patterns observed during development

Fluorochrome conjugated lectins were used to observe cell surface changes in the corneal endothelium during wound repair in the adult rat and during normal fetal development. Fluorescence microscopy of non-injured adult corneal endothelia incubated in wheat-germ agglutinin (WGA), Concanavalin A (Con A), and Ricinus communis agglutinin I (RCA), revealed that these lectins bound to cell surfaces. Conversely, binding was not observed for either Griffonia simplicifolia I (GS-I), soybean agglutinin (SBA) or Ulex europaeus agglutinin (UEA). Twenty-four hours after a circular freeze injury, endothelial cells surrounding the wound demonstrated decreased binding for WGA and Con A, whereas, RCA binding appeared reduced but centrally clustered on the apical cell surface. Furthermore, SBA now bound to endothelial cells adjacent to the wound area, but not to cells near the tissue periphery. Neither GS-I nor UEA exhibited any binding to injured tissue. By 48 h post-injury, the wound area repopulates and endothelial cells begin reestablishing the monolayer. These cells now exhibit increased binding for WGA, especially along regions of cell-to-cell contact, whereas, Con A, RCA and SBA binding patterns remain unchanged. Seventy-two hours after injury, the monolayer is well organized with WGA, Con A and RCA binding patterns becoming similar to those observed for non-injured tissue. However, at this time, SBA binding decreases dramatically. By 1 week post-injury, binding patterns for WGA, ConA and RCA closely resemble their non-injured counterparts while SBA continues to demonstrate low levels of binding. In early stages of its development, the endothelium actively proliferates and morphologically resembles adult tissue during wound repair.(ABSTRACT TRUNCATED AT 250 WORDS)