Postnatal reorganization of actin filaments and differentiation of intercellular boundaries in the rat aortic endothelial cells

Postnatal change in the distribution of actin filaments in endothelial cells was studied in the rat aorta by use of rhodamine-phalloidin staining and confocal laser scanning microscopy. Endothelial cells of the rat aorta possessed two populations of actin filament bundles, namely, peripheral bands at the cell border and stress fibers running longitudinally in the cytoplasm. Aortic endothelial cells of the neonatal rat contained only stress fibers, whereas those of the 10-day-old rat developed both peripheral bands and stress fibers. After 20 days of age, aortic endothelial cells had predominantly peripheral bands with occasional stress fibers around the branch orifices. During postnatal development the length density of stress fibers in aortic endothelial cells decreased, whereas individual stress fibers in endothelial cells were shortened. Electron-microscopic observation revealed that the high intercellular boundaries of aortic endothelial cells at birth decreased in height and developed cytoplasmic interdigitations after 20 days of age. The occurrence of peripheral bands at the cell border is thought to be closely related to formation of cytoplasmic interdigitation which strengthens the mechanical connection between endothelial cells against increasing transmural pressure. Expression of stress fibers in aortic endothelial cells of the neonatal rat is supposed to be affected by longitudinal elongation of the developing aorta, whereas their postnatal decrease is though to be correlated with the change of fluid shear stress loaded in the aortic endothelium.     

Reversible changes in stress fiber expression and cell shape in regenerating rat and rabbit aortic endothelium

The influence of intimal de-endothelialization on stress fiber expression in regenerating rat and rabbit aortic endothelium was studied using immunofluorescence microscopy. Rat thoracic and abdominal aortae were balloon de-endothelialized, and endothelial cell shape and stress fiber expression was studied in both uninjured and de-endothelialized animals. In control animals, the majority of thoracic endothelial cells did not contain stress fibers while the majority of abdominal endothelial cells did. One week after injury, all the endothelial cells distal to the regenerating edge contained very prominent stress fibers. In areas directly adjacent to the still de-endothelialized surface, the endothelial cells had an intense, diffuse cytoplasmic staining without stress fibers. Regenerating endothelium also had a substantially higher length-to-width ratio, but smaller cell areas. Six weeks after injury, the endothelium had completely regenerated, and stress fibers were lost from the majority of the thoracic endothelial cells. Changes in abdominal aorta stress fiber expression were not as marked. In the rabbit, all the control thoracic endothelial cells had stress fibers; however, cells at the leading edge of a narrow region of de-endothelialization had few stress fibers. The results suggest that stress fibers do not play a primary role in cellular migration in situ. The transient increase in stress fiber expression in the rat may result from a temporary demand for greater adhesive capabilities until the subendothelial extracellular matrix is remodeled.     

Developmental stage dependent expression of the endothelial stress fibers and organization of fibronectin fibrils in the aorta of chick embryos

Organizational relationships between endothelial stress fibers and fibronectin fibrils in the developing chick abdominal aorta, from 5th day embryos to 3rd day young chicks, were studied with immunofluorescence and electron microscopy. Stress fibers, axially aligned parallel to the longitudinal cell axis, were expressed in the largely elongated endothelial cells, in embryos older than 8th day of incubation. Fibronectin fibrils in the aortic basal lamina, changed its organizational pattern from the network-like form to the straight bundles arranged parallel to the vessel's longitudinal axis after 9th day of incubation. Such axial alignment was dominant in the matrix beneath the elongated cells containing stress fibers, suggesting the existence of stress fibers may possibly modify the fibronectin's organizational pattern. The vinculin-containing dense plaque, which shaped like as the adhesion plaque in the cultured cells, was located at the ends of or lateral associating sites of stress fibers in embryos older than 8th day stage. The expression of stress fibers, as well as the formation of stress fiber's end plaques, may closely relate to the alignment between the stress fiber and fibronectin fibrils in the extracellular matrix.     

Expression and intracellular distribution of stress fibers in aortic endothelium

Immunofluorescence microscopy was used to determine the number of endothelial cells with stress fibers for three age groups, and for three distinct anatomical locations within the descending thoracic aorta of both normotensive and spontaneously hypertensive rats. For each age group examined, hypertensive rats consistently demonstrated greater stress fiber expression than did normotensive rats. Neither age nor blood pressure was the predominant influence on stress fiber expression in aortic endothelium. In the normotensive rats, stress fiber expression remained unchanged for all age groups examined. For both strains, however, more endothelial cells with stress fibers were found in those regions where fluid shear stresses are expected to be high, when compared with those regions where the fluid shear stresses are expected to be low. This observation suggests that anatomical location, with its implied differences in fluid shear stress levels, is a major influence on stress fiber expression within this tissue. Electron microscopy was used to determine the intracellular distribution of stress fibers for both strains. Most stress fibers in both strains were located in the abluminal portion of the endothelial cells. This result is consistent with a role for stress fibers in cellular adhesion. However, the hypertensive rats had a higher proportion of stress fibers in the luminal portion of their cytoplasm than the normotensive rats. This increased presence of stress fibers in the luminal portion of the cell may be important in maintaining the structural integrity of the endothelial cell in the face of elevated hemodynamic forces in situ.     

In vitro angiogenesis in fibrin matrices containing fibronectin or hyaluronic acid.

Angiogenesis involved numerous interactions between extracellular matrix and endothelial cells which may exhibit changes in actin filament distribution. Using an in vitro model, capillary endothelial cells were grown in fibrin matrix containing fibronectin or hyaluronic acid. Actin filament distribution, nucleus localization and cell morphology were observed. Preliminary study showed the formation of tube-, branche- and capillary-like structures within fibrin. In the presence of both fibrin and fibronectin, cells with actin filament stress fibers were more spreading than those in fibrin. In the presence of hyaluronic acid, tubes were limited in extension into the fibrin. In addition, the study of co-localization of nucleus and actin filaments showed different cell behaviours. Migratory cells seem to arrange in parallel to each other and a capillary-like structure may be formed at the proximal extremity of this cell pattern.     

Immunohistochemical and histoenzymological characteristics of the arterial intimal cells in nonspecific aorto-arteritis

Enzymatic activity of cells, antigenic cellular markers and extracellular matrix of the hyperplastic intima of the aorta and carotid arteries was investigated in non-specific aorto-arteritis by immunomorphological and histochemical techniques. The cells of subendothelial layer of thickened arterial intima contained smooth muscle cell myosin, gave positive reactions to myosin ATP-ase and revealed high activity of thiamine pyrophosphatase. Fibronectin and type IV and V collagen were located in close proximity to these cells. The data obtained make it possible to consider these cells as modified smooth muscle cells. Type III collagen was the prevalent type of extracellular matrix of the thickened intima. A great number of blood vessels of the capillary and precapillary types have been found to penetrate into the intima from the adventitia. A possible role of pericytes surrounding newly formed capillaries as the precursors of subendothelial cell population in the hyperplastic intima is discussed.     

The circulating alpha 1-antitrypsin-elastase complex attacks the elastic lamina of blood vessels. An immunohistochemical study

The aim of the study was to determine the destination of the alpha 1-antitrypsin-elastase complex, which is found in circulating blood after the peroral administration of elastase. The complex was made in vitro by mixing hog pancreatic elastase with human alpha 1-antitrypsin and then injected intravenously into rats and mice. Tissues taken at various times after injection were subjected to histochemical staining using an antibody against elastase. Light microscope observations revealed dense deposition of reaction products in the elastic lamina of the arterioles; moderate or slight deposits were seen in the tissues surrounding arteries, in the tubular epithelial cells of the proximal convoluted tubules in the kidney, and in the pancreatic ducts. Immunoelectron microscopy revealed heavy deposition of the reaction product in the elastic lamina of the small arteries and arterioles; some dissolution of the elastic fibers was also evident. Pinocytic uptake of the alpha 1-antitrypsin-elastase complex was observed on the abluminal surface of endothelial cells and in smooth-muscle cells bordering the elastic lamina of arterioles. The endothelial cells of the arteries and arterioles retained their normal morphological appearance, although local desquamation was observed in some animals. The results indicate that, when the alpha 1-antitrypsin-elastase complex is present in the circulating blood, it is incorporated into the elastic lamina through the endothelial layer. This results in liquefaction of the lamina, desquamation of endothelial cells and leakage of the complex into the perivascular tissues via the vascular walls. However, some of the complex seems to be excreted very quickly from the kidneys.     

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.     

Diversity of vinculin/meta-vinculin in human tissues and cultivated cells. Expression of muscle specific variants of vinculin in human aorta smooth muscle cells

Microheterogeneity of different vinculin and meta-vinculin isoforms in adult human tissues and cultured cells was studied by two-dimensional gel electrophoresis and immunoblotting technique. Four isoforms of vinculin (alpha, alpha', beta, and gamma) and two isoforms of meta-vinculin (alpha and beta) were resolved. alpha-, alpha'-, and beta-isoforms of vinculin were found in all cell types and tissue samples analyzed in the present study. gamma-Isoform of vinculin and both alpha- and beta-isoforms of meta-vinculin were found in smooth (aorta wall and myometrium) and cardiac muscle, rather than in skeletal muscle, liver, foreskin fibroblasts, and macrophages. In the primary culture of human aorta smooth muscle cells, the fractional content of gamma-isoform of vinculin and meta-vinculin was dramatically reduced, and, by the onset of intensive cell division, the proteins could hardly be detected. Subcultured human aorta smooth muscle cells did not contain gamma-vinculin and meta-vinculin. We analyzed the microheterogeneity of vinculin and meta-vinculin in three smooth muscle layers of human aorta wall--media, muscular-elastic (adjacent to media) intima, and subendothelial (juxtaluminal) intima. It was shown that in media the fractional content of gamma-isoform of vinculin was 45% and meta-vinculin, 42%; in muscular-elastic intima the fractional content of gamma-vinculin was 42% and meta-vinculin, 36%. However, in subendothelial intima, the share of these proteins was significantly lower than in adjacent muscular-elastic intima and media. Isoactin pattern that is characteristic of smooth muscle was identical in all aortic layers, thus proving the smooth muscle origin of subendothelial intima cells. These findings demonstrate that human aortic smooth muscle cells in vivo and in vitro undergo coordinated differential expression of smooth muscle specific variants of vinculin, i.e. gamma-vinculin and meta-vinculin.     

A new view of convective-diffusive transport processes in the arterial intima

In this paper a new theoretical framework is presented for analyzing the filtration and macromolecular convective-diffusive transport processes in the intimal region of an artery wall with widely dispersed macromolecular cellular leakage sites, as proposed in the leaky junction-cell turnover hypothesis of Weinbaum et al. In contrast to existing convection-diffusive models, which assume that the transport is either 1-D, or convection is primarily in a direction normal to the endothelial surface, the present model considers for the first time the nonuniform subendothelial pressure field that arises from the different hydraulic resistances of normal and leaky endothelial clefts and the special role of the internal elastic lamina (IEL) in modulating the horizontal transport of macromolecules after they have passed through the leaky clefts of cells that are either in mitosis or demonstrate IgG labeling. The new theory is able to quantitatively explain the growing body of recent experiments in which an unexpectedly rapid early-time growth of the leakage spot has been observed and the longer time asymptotic behavior in which the leakage spot appears to approach an equilibrium diameter. The new theory also predicts the observed doubling in macromolecular permeability between EBA labeled blue and white areas when the frequency of leakage sites is doubled. This frequency for doubling of permeability, however, is an order of magnitude smaller than predicted by the author's previous model, Tzeghai et al., in which only convection normal to the endothelial surface was considered and the pressure was uniform in the intima. The longer time model predictions are used to explain the time scale for the formation of liposomes in subendothelial tissue matrix in animal feeding experiments where it has been observed that the extracellular lipid concentration rises sharply prior to the entry of monocytes into the intima.     

Localization of nuclear matrix core filament proteins at interphase and mitosis.

The gentle removal of chromatin uncovers a nuclear matrix consisting of two parts: a nuclear lamina connected to the intermediate filaments of the cytoskeleton and an internal matrix of thick, polymorphic fibers connecting the lamina to masses in the nuclear interior. This internal nuclear matrix can be further fractionated to uncover a highly branched network of 9 nm and 13 nm core filaments retaining some enmeshed bodies. The core filament network retains most of the nuclear RNA, as well as the fA12RNP antigen, and may be the most basic or core element of internal nuclear structure. One high molecular weight protein component of the core filament network, the H1B2 antigen, is normally masked in the interphase nucleus and is uncovered as the chromatin condenses at mitosis. This protein is associated with a fibrogranular network surrounding and connected to the chromosomes. The core filament-associated fA12 antigen also becomes associated with this perichromosomal network. We propose that the core filament nuclear matrix structure may not completely disassemble at mitosis but, rather, that parts remain as a structural network connected to chromosomes and other mitotic structures. These mitotic networks may, in turn, serve as the core structures on which the nuclear matrices of daughter cells are built.     

Ultrastructural and immunohistochemical study of experimental atherosclerosis in Japanese quails

A marked plaque was produced at the tunica intima of the ascending aorta in all of the Japanese quails of 9 weeks old which fed on atherogenic diet containing 2% cholesterol for 8 weeks, while no structural changes of aortic wall were observed in Japanese quails which fed on normal basic food for the same period. The media of aorta in normal quails consist of smooth muscle cells, myofibroblast-like cells (MF) cells), and many successive elastic membranes. At the atherosclerotic lesion, many MF cells migrated from media into intima, and a part of smooth muscle cells were also differentiated to MF cells. Moreover, the most migrating MF cells differentiated to foam cells at the intimal thickness regions, and a few other MF cells also differentiated into endothelial cells of newly forming capillaries. By the immunohistochemical stainings, medial smooth muscle cells were negatively stained with anti-vimentin antibody, and the majority of cells in the intima (MF cells, foam cells, and endothelial cells) contained vimentin filaments. These results indicate that MF cells play a very important role in the development of atherosclerosis in Japanese quail. The morphologicals study offers some new insights into the evaluation of Japanese quails as an animal model of atherosclerosis.     

Stress fibers in the splenic sinus endothelium in situ: molecular structure, relationship to the extracellular matrix, and contractility

In the present study, we investigated structural and functional aspects of stress fibers in a cell type in situ, i.e., the sinus endothelium of the human spleen. In this cell type, stress fibers extend underneath the basal plasma membrane and are arranged parallel to the cellular long axis. Ultrastructurally, the stress fibers were found to be composed of thin actin-like filaments (5-8 nm) and thick myosin-like filaments (10-15 nm X 300 nm). Actin filaments displayed changes in polarity (determined by S-1-myosin subfragment decoration), which may allow a sliding filament mechanism. At their plasmalemmal attachment sites, actin filaments exhibited uniform polarity with the S-1-arrowhead complexes pointing away from the plasma membrane. Fluorescence microscopy showed that the stress fibers have a high affinity for phalloidin and antibodies to actin, myosin, tropomyosin, and alpha-actinin. Vinculin was confined to the cytoplasmic aspect of the plasmalemmal termination sites of stress fibers, while laminin, fibronectin, and collagens were located at the extracellular aspect of these stress fiber-membrane associations. Western blot analysis revealed polypeptide bands that contained actin, myosin, and alpha-actinin to be major components of isolated cells. Exposure of permeabilized cells to MgATP results in prominent changes in cellular shape caused by stress fiber contraction. It is concluded that the stress fibers in situ anchored to cell-to-extracellular matrix contacts can create tension that might allow the endothelium to resist the fluid shear forces of blood flow.     

Localization of proteoheparan sulfate in rat aorta

This study describes the distribution of heparan sulfate proteoglycan ( HSPG ) within the rat aorta using immunocytochemical (biotin-avidin-peroxidase) and immuno-electron microscopy (125I-autoradiography). Heparan sulfate proteoglycan was isolated from a basement membrane producing mouse EHS sarcoma ( Hassell et al. 1980) and used to generate antisera in rabbits. Light microscopic observations revealed intense immunostaining of the intima and media of normal aorta, adventitial vasa vasorum, and aortic intimal fibromuscular thickenings induced by experimental injury (balloon de-endothelialization). Immunoelectron microscopy using 125I labeled antibodies to HSPG revealed that proteoheparan sulfate was localized to the amorphous layer of basement membrane below aortic and capillary endothelium. In addition, labeled anti- HSPG could be localized to the external lamina surrounding the smooth muscle cells in the hyperplastic intima. These studies reveal that antibodies prepared against a proteoheparan sulfate isolated from a basement membrane producing EHS sarcoma cross react with basement membrane structures within the aortic wall. Furthermore, these results demonstrate that the basement membranes beneath aortic and capillary endothelium and the external lamina surrounding aortic smooth muscle cells contain a heparan sulfate proteoglycan that is antigenically similar.     

The three-dimensional structure of the tunica intima and tunica media of the human fetal aorta studied by a new method]

A new method is developed for revealing the latent surfaces in the structure of organs by scanning electronic microscopy. The method is based on the treatment of specimens with potassium ethoxide until cells start to appear in the dissociating solution. Using this method, thoracic aorta of nine human fetuses at the stage of 20-28 weeks was studied. Subendothelial intima and media of human fetal aorta contain smooth muscle cells differing by their arrangement, shape and surface microrelief. The intima cells are arranged in a mosaic pattern formed of single cells or cell clusters. By means of cell processes they are connected with each other, as well as with endothelial and smooth muscle cells of the media. Smooth muscle cells in the inner part of the media also have processes and form an open network. Part of the cells penetrate the intima through pores of the inner elastic membrane. In the deeper layers of the media, laterally adjoining spindle-shaped smooth muscle cells are found. It is suggested that the observed cell polymorphism is due mostly to penetration of the media smooth muscle cells into subendothelium and modification of their shape under the effect of the microenvironment.     

Demonstration of fibronectin in normal and injured aorta by an indirect immunoperoxidase technique

The presence and localization of fibronectin in normal and mechanically injured aorta in rabbits was studied using an indirect immunoperoxidase technique on tissue specimens fixed in formaldehyde, embedded in paraffin and pretreated with pepsin. The effect on staining quality of treatment with testicular hyaluronidase prior to immunoperoxidase staining was also examined. In the intima from normal aorta fibronectin was present in the subendothelial basal layer, along the internal and external elastic laminae, around and between the smooth muscle cells of the media and along the collagen and elastic fibres in the adventitia. Sixteen days after a single mechanical dilatation of the descending thoracic aorta all animals developed gross atherosclerotic-like changes. Microscopic examination revealed prominent neo-intimal hyperplasia with subendothelial, cushion-like thickenings but no medial or adventitial alterations. Fibronectin, in increased amounts, was found between and around the endothelial cells and in the subendothelial thickenings between the proliferating smooth muscle cells in relation to the fine, thin elastic and argyrophilic fibres. In the media and adventitia the amount and distribution of fibronectin was indistinguishable from uninjured control aortas. Treatment with testicular hyaluronidase before immunoperoxidase staining resulted in a higher staining resolution in normal and injured aorta. The conspicuous observation in the present study is that fibronectin exclusively accumulates in areas of tissue repair. The origins and functions of fibronectin during tissue injury and repair are discussed.     

Localization of proteoheparan sulfate in rat aorta

Summary This study describes the distribution of heparan sulfate proteoglycan (HSPG) within the rat aorta using immunocytochemical (biotin-avidin-peroxidase) and immunoelectron microscopy (¹²⁵I-autoradiography). Heparan sulfate proteoglycan was isolated from a basement membrane producing mouse EHS sarcoma (Hassell et al. 1980) and used to generate antisera in rabbits. Light microscopic observations revealed intense immunostaining of the intima and media of normal aorta, adventitial vasa vasorum, and aortic intimal fibromuscular thickenings induced by experimental injury (balloon de-endothelialization). Immunoelectron microscopy using ¹²⁵I labeled antibodies to HSPG revealed that proteoheparan sulfate was localized to the amorphous layer of basement membrane below aortic and capillary endothelium. In addition, labeled anti-HSPG could be localized to the external lamina surrounding the smooth muscle cells in the hyperplastic intima. These studies reveal that antibodies prepared against a proteoheparan sulfate isolated from a basement membrane producting EHS sarcoma cross react with basement membrane structures within the aortic wall. Furthermore, these results demonstrate that the basement membranes beneath aortic and capillary endothelium and the external lamina surrounding aortic smooth muscle cells contain a heparan sulfate proteoglycan that is antigenically similar.     

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.     

An ultrastructural study of centriolar complexes in adult and embryonic human aortic endothelial cells

Ultrastructural organization of centriolar complexes in 90 adult human aortic endothelial cells from uninvolved areas, fibrous and atheromatous plaques and 30 endothelial cells from human embryonic aorta were studied using serial sections. Primary cilia protruding from the abluminal cell surface were found on 28 of 30 endothelial cells from atheromatous plaques. Only five of 30 cells from either fibrous plaques or uninvolved areas developed primary cilia protruding to the lumen. Impaired primary cilia entirely immersed into the cytoplasm were found in embryonic endothelial cells. It was speculated that both the modes of formation and the functions of endothelial cilia in embryonic and adult aortas are different.     

Basal lamina formation by cultured microvascular endothelial cells

The production of a basal lamina by microvascular endothelial cells (MEC) cultured on various substrata was examined. MEC were isolated from human dermis and plated on plastic dishes coated with fibronectin, or cell-free extracellular matrices elaborated by fibroblasts, smooth muscle cells, corneal endothelial cells, or PF HR9 endodermal cells. Examination of cultures by electron microscopy at selected intervals after plating revealed that on most substrates the MEC produced an extracellular matrix at the basal surface that was discontinuous, multilayered, and polymorphous. Immunocytochemical studies demonstrated that the MEC synthesize and deposit both type IV collagen and laminin into the subendothelial matrix. When cultured on matrices produced by the PF HR9 endodermal cells MEC deposit a subendothelial matrix that was present as a uniform sheet which usually exhibited lamina rara- and lamina densa-like regions. The results indicate that under the appropriate conditions, human MEC elaborate a basal lamina-like matrix that is ultrastructurally similar to basal lamina formed in vivo, which suggests that this experimental system may be a useful model for studies of basal lamina formation and metabolism.