人胚胎冠状动脉组织发生的光镜研究

本文用Van Gieson-orcein染色法对35例(第5周至第10个月)人胚胎冠状动脉的组织发生作了光镜观察.结果显示第5周心外膜内的间充质细胞形成了血管壁.第6周冠状动脉初步形成,内皮直接贴附于内弹性膜,中膜为1～2层平滑肌细胞,外弹性膜为2层弹性纤维组成,外膜厚于中膜.内皮下层第6个月可辨认出来.内弹性膜、中膜、外膜的厚度及管径均随胎龄而增加.内弹性膜厚度随胎龄呈直线上升,中膜和外膜厚度随胎龄呈指数曲线上升.内径和外径的比值在各期胎龄保持相对衡定.     

An autoradiographic technique for en-face preparations of aortic endothelium

A method for the en-face preparation of aortic endothelium was developed from various different published accounts of the technique. This was adapted for autoradiography. Eighteen mice had tritiated thymidine (1 microCi/g body weight) injected and were killed 1 day later. Perfusion fixed aortae were cut into segments and placed face down on gelatin-coated slides. The arterial media and adventitia were removed and the en-face preparations coated with autoradiographic stripping film. After an exposure of 12 weeks the autoradiographs were developed and stained with Harris' haematoxylin. Labelled endothelial cells were clearly distinguishable and there was no distortion of the cells compared with other en-face and whole mount preparations. The technique described is simple, reliable and produces consistent results.     

The study of wall deformation and flow distribution with transmural pressure by three-dimensional model of thoracic aorta wall

The sensitivity of shear stress over smooth muscle cells (SMCs) to the deformability of media layer due to pressure is investigated in thoracic aorta wall using three-dimensional simulations. A biphasic, anisotropic model assuming the radius, thickness, and hydraulic conductivity of vessel wall as functions of transmural pressure is employed in numerical simulations. The leakage of interstitial fluid from intima to media layer is only possible through fenestral pores on the internal elastic lamina (IEL). The media layer is assumed a heterogeneous medium containing SMCs embedded in a porous extracellular matrix of elastin, proteoglycan, and collagen fibers. The applicable pressures for the deformation of media layer are varied from 0 to 180 mmHg. The SMCs are cylindrical objects of circular cross section at zero pressure. The cross sectional shape of SMCs changes from circle to ellipse as the media is compressed. The local shear stress over the nearest SMC to the IEL profoundly depends on pressure, SMCs configurations, and the corresponding distance to the IEL. The consideration of various SMC configurations, namely the staggered and square arrays, mimics various physiological conditions that can happen in positioning of an SMC. The results of our simulations show that even the second nearest SMCs to the IEL can significantly change their functions due to high shear stress levels. This is in contrast to earlier studies suggesting the highest vulnerability to shear stress for the innermost layer of SMCs at the intimal-medial interface.     

Ontogeny of vessel wall components in the outflow tract of the chick

During development of the outflow tract, the walls of the truncus arteriosus change from a diffuse extracellular matrix (ECM) surrounded by an extension of the myocardium to alternating laminae of smooth muscle and elastic connective tissue. The transition rapidly follows septation, when mesenchyme associated with the endothelium differentiates. Using immunocytochemical methods with antibodies to components of the tunica media and the tunica adventitia we have analysed the differentiation of the vessel walls of the outflow tract of the chick. The tunica media marker, elastin, forms laminae in a radial sequence, beginning at the outer margin of the truncus mesenchyme. Conversely, smooth muscle myosin is first expressed in cells associated with the endothelium. Laminin is expressed as a cell surface component throughout the development of the outflow tract. Matrix fibronectin distribution is correlated with the regions that will form the tunica media and apparently forms a radial gradient which is highest near the endothelium. Markers for the tunica adventitia, collagen I and VI, are expressed first at the peripheries of the newly formed tunica media, and collagen VI expression spreads radially through the tunica media. Thus, the vessel wall components appear within the mesenchyme of the truncus arteriosus in opposed radial gradients of differentiation. The tunica media cells acquire secretory and contractile phenotypes independently and may be responding to different stimuli in their expression of these features.     

Reversible microcarrier-mediated junctional communication between endothelial and smooth muscle cell monolayers: an in vitro model of vascular cell interactions

Junctional communication between cultured monolayers of aortic endothelial and smooth muscle cells was established as an in vitro model of vessel wall cell interactions. Confluent monolayers of endothelial cells on microcarriers placed on the surface of a smooth muscle cell monolayer became attached within 1 hour. After 3 hours of contact co-culture, approximately 4% of each monolayer was involved in heterocellular attachment (range 2% to 6%; 4 to 12 cells per microcarrier). Endothelial-smooth muscle cell junctions formed between the two cell populations at the sites of attachment on the lower surface of each microcarrier. When either endothelial cells or smooth muscle cells were prelabeled with [3H]uridine, intracellular nucleotide was rapidly transferred across the region of heterocellular attachment to the complementary cell population. Heterocellular gap junctional transfer was inhibited when endothelial-smooth muscle attachment was prevented by slowly rocking the culture vessel. No evidence of nucleotide transfer was obtained when endothelial cells were co-cultured with MDCK cells incapable of forming gap junctions. Contact co-culture of endothelium and smooth muscle cells were reversed by gentle agitation of the cultures; the microcarriers detached allowing the recovery of pure cell populations. The contact co-culture technique is well suited to studies of vascular cell interactions and is of general applicability to anchorage dependent cells.     

Structure and distribution of the lymphatic vessels in the parietal pleura of the monkey as studied by enzyme-histochemistry and by light and electron microscopy.

The entire distribution of lymphatics in whole mount preparations of the Japanese monkey was studied using the enzyme-histochemical technique reported by KATO et al. (1990, 1991). In this staining, the lymphatic endothelium was colored dark brown by its positive 5'-nucleotidase activity, while most blood vessels (especially arterioles) were colored blue due to their positive alkaline phosphatase reaction. The whole mount preparations of the pleura treated enzyme-histochemically clearly indicated the distribution, branching patterns and running courses of lymphatic vessels. They revealed numerous short blind-ending knobs which represented the initial portions of lymphatics. These knobs were seen near the surface of the parietal pleura along its entire extent. In the costal and diaphragmatic pleura, the lymphatics ran parallel to the intercostal muscle fibers, but perpendicular to the tendinous and muscular fibers of the diaphragm; they formed ladders, independent of the courses of blood vessels. In the mediastinal pleura, lymphatic vessels showed a tree-like branching accompanying blood vessels. Under the light microscope, toluidine-blue stained semithin sections revealed the initial part of lymphatics as a small irregularly outlined cavity (7-10 microns in diameter) surrounded by a dense connective tissue. This lymphatic dilation was sometimes located close to a thin mesothelial layer. Such a structure suggesting a "stoma" was seen near the attachment of the muscular diaphragm to the sternum and along the borders of the ribs. Transmission electron microscopy revealed an occasional interruption in the mesothelium. This stoma continued to a submesothelial cavity whose base comprised an attenuated endothelium of an extended lymphatic vessel.     

Smooth muscle cells and fibroblasts of the coronary arteries derive from epithelial-mesenchymal transformation of the epicardium

Previous research has revealed that cells contributing to coronary vascular formation are derived from the dorsal mesocardium, however, the fate of these cells during consecutive stages of heart development is still unclear. We have conducted a study regarding the recruitment of vascular components and the subsequent differentiation into mature vessel wall structures with the aid of immunohistochemical markers directed against endothelium, smooth muscle cells, and fibroblasts. The proepicardial organ including an adhering piece of primordial liver of quail embryos (ranging from HH15 to HH18) was transplanted into the pericardial cavity of chicken embryos (ranging from HH15 to HH18). The chicken-quail chimeras (n=16) were harvested from the early stage of endothelial tube formation (HH25) to the late stage of mature vessel wall composition (HH43). Before HH32 endothelial cells have invaded the myocardium to give rise to yet undifferentiated coronary vessels. These endothelial cells are not accompanied by other non-endothelial cells. The superficial epicardial layer changes from a squamous mesothelium into a cuboid epithelium preceding media and adventitia formation. Subsequently, a condensed area of mesenchymal cells delaminates from the cuboidal lining extending toward the vessel plexus. Around the coronary arteries, these mesenchymal cells differentiate into smooth muscle cells or fibroblasts as shown by immunohistochemical markers. We conclude that epithelial-mesenchymal transformation of the epicardial lining delivers the smooth muscle cells and fibroblasts of the coronary arterial vessel wall. Molecules involved in epithelial transformation processes elsewhere in the embryo are also expressed within the subepicardial layer, and are considered to participate in inducing this process.     

En face organ culture of vascular endothelium.

A simple technique is described for maintaining in organ culture segments of blood vessel in such a way that the endothelium can be studied en face. The behaviour of such cultures throws further light on the interaction between endothelium and intravascular clotting.     

The aortic intima in organ culture. Response to culture conditions and partial endothelial denudation.

A culture technique for whole blood vessel wall that preserves an intact and regenerative endothelium has been developed. The retention of an intact endothelium allows careful observation of the tissue as it responds to culture conditions, responses that include a change of replication timing and the induction of particulate endocytosis. Complete and normal regeneration of the endothelium in explants has made possible evaluation of the differences between the regeneration of endothelial cell monolayers and regeneration of intima in vivo. From these comparisons it appears that the shorter induction time of endothelial migration and proliferation and the more rapid migration in endothelial cell monolayers are due to effects of the culture medium or plastic culture surface, while the higher than normal cell density found in the intima after wound recovery in vivo probably is due to the dynamics of the blood vessel itself. The ability to control cell-cell interactions on the cultured tissue has made possible the investigation of gap junction-mediated metabolic interactions in regenerating intima. Results indicate that the disruptive effects of intimal regeneration do not depend on or produce an obvious change in junction-mediated nucleotide transfer between endothelial cells.     

A method of preparing slide samples of human arterial endothelium impregnated with silver

The paper deals with the preparation of endothelial monolayers "en face". New approach is based on the silver impregnation of cell borders prior to the endothelium replacing from the vascular wall.     

Morphology of the carotid sinus wall in normotensive and spontaneously hypertensive rats

The morphology of the carotid sinus region of the internal carotid artery was studied in spontaneously hypertensive rats (SHR) at 5, 8, 16, and 24 weeks of age. The carotid sinus region occupied the proximal millimeter of the internal carotid artery, and was easily recognizable by the presence of an extensive adventitial capillary plexus, which was absent on adjacent arteries (e.g., common and external carotid arteries). Methylene blue-stained whole-mount preparations showed the extent of baroreceptor nerves over the sinus. Baroreceptor fibers terminated in distinctive bulbous-like endings, which, at the ultrastructural level, were filled with mitochondria. No differences were noted in the sinus adventitial capillary network or baroreceptor distribution between SHR and age-matched Wistar-Kyoto (WKY) normotensive control animals. With the onset of a significant rise in SHR blood pressure, the carotid sinus wall increased in thickness and total vessel size. The wall/lumen ratios were significantly larger in the SHR than in age-matched WKY ratios in all age groups. SHR carotid sinus vessel enlargement was uniform throughout the vessel tunics, with no significant change in the proportion of the tunica media occupied by smooth muscle cells. The increase in the carotid sinus wall thickness associated with increasing hypertension could affect the ability of the sinus to distend and may play a secondary role in the maintenance of hypertension by compromising baroreceptor nerve ending sensitivity.     

A method for the preparation of imprints from the nasal mucosa

Important immunological reactions take place on the surface of mucosal membranes. Improved methods for the sampling and quantitative study of the cells taking part in these reactions are therefore desirable. We here describe a new technique for the preparation of imprints from the nasal mucosa. The method utilizes a plastic film coated with a thin layer of an albumin-glycerol mixture to improve cell adherence to the surface. The membrane is gently pressed onto a defined portion of the mucous membrane. Fixation and staining procedures are performed on the plastic film, which is then mounted on a slide and covered by a coverslip. The preparations have excellent optical properties and specific cell types can be easily studied, quantified and related to the specific area of the mucosa from which the imprint was taken.     

Melanoma cell adhesion to injured arterioles: mechanisms of stabilized tethering

An isolated perfused vessel model was used to examine the mechanisms underlying the adhesive interactions between circulating tumor cells and subendothelial matrix in denuded arterioles. Arterioles ranging from 70 to 100mm in diameter were isolated from rat mesentery, transferred to an isolated vessel chamber, cannulated on both ends with glass micropipettes, and perfused with media containing 106 hamster melanoma (RPMI 1856) cells/ml. In a second group of arterioles, the endothelium was denuded by running 2ml of air through the vessel lumen. Since the tumor cells did not adhere to the vessel wall when perfused at physiologically relevant shear rates, perfusate flow was stopped and the tumor cells were allowed to settle onto the vessel wall for 20 min. After counting the number of tumor cells that settled onto the arteriolar wall, perfusate flow was re-initiated and unattached cells were washed away. The number of cells remaining adherent were counted and the percentage of adherent cells (relative to the total number of cells that settled on to the vessel wall during the period of no-flow) were calculated and compared among different groups. We observed that tumor cells are much more adhesive to denuded arterioles than to intact arterioles. To determine the mechanisms responsible for the adhesive interactions that become established and stabilized during the period of flow reduction, denuded arterioles were treated with fibronectin antiserum or Arg-Gly-Asp (RGD) peptides. Both treatments significantly reduced tumor cell adhesion to denuded arterioles. In subsequent studies, melanoma cells were treated with a transglutaminase inhibitor, monodansylcadaverine (MDC), which reduced the ability of adherent tumor cells to withstand the anti-adhesive effects of a subsequent increase in perfusate flow rate after the period of no-flow. Our data suggest that tumor cells adhere to fibronectin in the subendothelial matrix in denuded arterioles by an RGD-dependent mechanism. Moreover, our observations are consistent with the concept that a transglutaminase-catalysed reaction acts to stabilize the adhesive interactions between subendothelial matrix components and melanoma cells during the period of flow stasis such that the cells are able to withstand subsequent substantial increases in wall shear rate and remain adherent.     

The relationship of the macrophage system to the blood vessel wall and atherogenesis

Monocytes/macrophages are physiological cellular components of the subendothelium of greater arteries as has been shown by our studies on endothelial/intimal preparations of 9 species, including man. Differentiation is possible between species rich (pig, man, sheep) or poor in macrophages (rabbit and other small laboratory animals) according to their density of subendothelial infiltration in the aorta. Morphological, histochemical, and ultrastructural results have shown that we are mainly dealing with metabolic quiescent, resident macrophages, their number being regulated by influences depending on endothelium and intima factors. We assume that in species rich in macrophages and in man, they have physiological functions for homoeostasis of border layer and intima. The most considerable signal for subendothelial macrophage accumulation in man, pig, and rabbit is intimal deposition of lipids, independent of experimental or spontaneous induction. Short-term or long-term hypercholesterolemia is not associated with increased adhesion and emigration of monocytes in unchanged intima at venous or arterial endothelial cells of pigs and rabbits. There is no evidence to intimal lipid infiltration being preceded by subendothelial macrophage invasion. Most studies on vessel border layer have suggested that in the early high-lipid phase of atherogenesis subendothelial macrophages participate in lipid metabolic clearance. But in conditions of metabolic activation they can be an atherogenic danger due to a whole range of secretory products stimulating recruitment of blood monocytes and lymphocytes, increasing the permeability of endothelium, altering the extracellular matrix components, and causing modulation of gen expression of vessel wall cells. Therefore, metabolic activation of the subendothelial macrophage system is a new, potentially atherogenic mechanism.     

X-ray contrast media induce aortic endothelial damage, which can be prevented with prior heparin treatment

X-ray contrast media induce damage to the endothelial layer of vessels and initiate the formation of thrombosis, which is a complication for clinical diagnostic procedures. The future determination of the mechanisms, which underlie the damaging effect of X-ray contrast medium on vascular wall cells, especially vascular endothelium and possible prevention of this damage by vasoprotector, will result in a larger application in diagnostic procedures. The aim of the present study is to analyze the effect of X-ray contrast media (Verographin, Iodamid and Iodolipol) on the arterial endothelium morphology by using ultrastructural techniques (scanning and transmission electron microscopy, SEM and TEM respectively). Experiments have been carried out on New Zealand white rabbits (6 month old) and Wistar rats (6-8 month old) after a single injection of X-ray contrast media with and without prior heparin treatment. Control groups of animals were exposed to the same procedure but without X-ray contrast media injection and only received isotonic saline solution. The following time points were selected: 1, 6, 24, 72 h and 7 days. At the end of the experiments, animals were anesthetized by pentobarbital and then perfused with a balanced buffer for 1 min and followed by perfusion fixation with Karnovsky's fixative containing buffered solution of 2.5% paraformaldehyde and 2.5% glutaraldehyde (pH 7.36) at least 30 min. The aortic tissue was removed and immediately placed into a fresh portion of the same fixative. Aortic samples were then prepared for scanning and transmission electron microscopy (SEM and TEM respectively). Immediately after the injection of X-ray contrast media, the number of microvilli and blebs on the luminal surface of the endothelial cells (EC) significantly increased. Very often, nuclear portions of the EC sharply protruded into the vessel lumen. Clusters of spindle-shaped EC were seen throughout the endothelial monolayer. These changes persist through the 72-h period after X-ray contrast media injection. Moreover, the desquamation and denudation of the EC from the monolayer is often observed and this is accompanied by the presence of a microthrombus on the vessel surface. Seven days after the post-injection period, endothelial monolayers still show severe damage, which often coexists with the presence of a different sized microthrombus on the vessel surface. However, the degree of lesion formation in most areas is substantially decreased as compared to the early period of post-injection (24 and 72 h). Heparin treated group shows intact morphology similar to the control experimental groups (saline injected group). Infrequently, minimal morphological changes of the endothelium, such as increased number of microblebs and microvilli, were seen with heparin treatment. We conclude that the negative side effects of the X-ray contrast media can be eliminated by a single injection of heparin or other vasoprotector prior to the diagnostic procedure.     

In vivo measurement of blood vessel wall thickness.

To understand the mechanical properties of arteries and vascular grafts, it is crucial that the wall thickness in these vessels be known. Unfortunately, all availble methods for measuring this parameter require the removal of the vessel, which precludes the study of such vessels as a function of time. A new radiographic technique for measuring the wall thickness of arteries and vascular grafts in vivo, utilizing contrast materials injected into the vessel lumen and applied to the outer surface of the vessel, is described. Radiographs are obtained with a portable X-ray machine and analyzed using a calibrated microscope. The technique has been successfully applied to the in vivo measurement of wall thickness in canine arteries, veins, and experimental vascular grafts. It is concluded that the method provides better than 95% accuracy in a variety of vessels and that it can be used to study changes in vascular grafts after their implantation into the arterial circulation.     

A rapid method for the detection of early stages of atherosclerotic lesion formation.

A simple, rapid technique for detecting early changes in the arterial vessels of rats and rabbits fed an atherogenic diet is described. After perfusion fixation, the descending thoracic aorta was cytochemically stained with oil red O to detect intracellular lipid and with Hoechst 33342 dye to detect nuclear DNA. The vessels were whole mounted and the luminal surface examined en face using both transmitted light and epifluorescence microscopy. With this technique it is possible to identify and quantitate mononuclear cells adhering to the vessel wall, determine the distribution and number of intimal foam cells within the intima, and determine the mitotic index of the endothelium. Tissue samples can be quickly prepared using this technique, thus allowing rapid analysis of the influence of various substances on the early stages of atherosclerotic lesion formation in animals fed an atherogenic diet.     

Pre-eclampsia associated alterations of the elastic fibre system in umbilical cord vessels

Although pre-eclampsia (PE) is often associated with fetal hypoxia, hypertension and/or disturbed function of the fetal circulation, the effect of these altered hemodynamic parameters on the structure and composition of umbilical vessels has not been systematically investigated before. Therefore, this study focuses on PE-associated changes of the elastic fibre system in umbilical cord vessels investigated by light and electron microscopy, immunocytochemistry and biochemistry. In umbilical cord veins, no changes in thickness of the vessel wall or of any sublayer were observed. However, the internal elastic lamina of the veins was split in 80% of the PE-group in contrast to 20% in uncomplicated pregnancies. This effect was significant (α <0.01) from 36 weeks of gestation onwards. In umbilical cord arteries, the entire arterial vessel wall was found to be 15% thicker in PE than in uncomplicated pregnancies. The enlargement was caused by an increase of both the tunica intima and tunica media. The thickening of the tunica intima was attributed to a migration of smooth muscle cells towards the endothelium, accompanied by a splitting of the internal elastic lamina. Compared to uncomplicated pregnancies, smooth muscle cells of arteries and veins in PE showed a metabolic activation demonstrated by highly dilated endoplasmic reticulum. A semiquantitative score method as well as a quantitative dot blot assay indicated a PE-associated reduction of elastin expression in the arterial vessel walls. In summary, PE obviously induces a decrease of the elastin content accompanied by a thickening of the vessel wall in umbilical cord arteries. This remodeling of the elastic fibre system, together with an increased migration of smooth muscle cells, might represent part of the functional adaptation system of the umbilical cord arteries on the altered hemodynamic conditions in PE. Accepted: 29 November 1999     

Fine structure of the lymphatic capillary and the adjoining connective tissue area

The lymphatic capillaries possess salient morphological features which differentiate them from the blood capillaries. Instead of a definitive basement lamina surrounding the lymphatic wall, there are numerous fine filaments (about 60 A in diameter) that are attached to the lymphatic endothelia at areas of increased electron density that are similar to half desmosomes. The filaments extend for varying vessel to the surrounding connective tissue. Endothelial projections occur along both the luminal and abluminal surfaces of the cell. The intercellular junctions are varied in structure along the lymphatic wall. Terminal processes of adjacent endothelial apposed without overlapping. At various points along the intercellular junction the apposed surface membranes are held in close apposition by adhesion devices, (a) zonula occludens, (b) zonula adhaerens, and (c) macula adhaerens (desmosome). Frequently open junctions are observed along the lymphatic wall. It is suggested that the areas between apposing membrane surfaces which lack attachment devices may represent specialized regions along the lymphatic wall that are easily separated and would permit the passage of excess tissue fluids and large molecules into the lumen of the lymphatic vessel. The transport of colloidal carbon particles across the endothelium in vesicles of various sizes and via intercellular junctions is demonstrated.     

Application of virtual microscopy in clinical cytopathology.

Virtual microscopy (VM) refers to the use of an automated microscope and digital imaging technology to scan, store, and view glass slides. VM systems allow the user to view a scanned image of the entire slide at multiple magnifications on a computer screen. We tested VM to evaluate its possible utility in diagnostic cytopathology. Ten cervical-vaginal monolayered preparations (AutoCyte preparation) were scanned using a BLISS (Bacus Laboratories Inc. Slide Scanner) system. Approximately 20-30% of the cellular area of each slide was imaged. The cases were randomly chosen to include examples ranging from benign cellular changes (BCC) to high-grade squamous intraepithelial lesions (HSIL). The computer performed image tiling and fusing of multiple JPEG images to create a high-quality VM slide. Six examiners (two each of cytopathologists, senior residents, and cytotechnologists) blindly evaluated the VM slides using an image server program (WebSlide Browser thin client software). The cytopathologic diagnoses made on the VM slide were then compared to the original glass slide diagnoses. BLISS took 36-100 min (avg. 58.4 min) to scan the selected fields in a glass slide with file sizes ranging from 23.1-83.6 MB. Time taken by the examiners to render a diagnosis ranged from 1-15 min (avg. 4.1 min) per case. The combined diagnostic accuracy was 98.3%. Only one case of LSIL was missed by one examiner. VM is a promising new tool, which gives a user the feel and simulated experience of an actual microscopic examination and provides a useful alternative to a glass slide in diagnostic cytopathology. Possible applications include: 1) second opinion consultation without transporting the glass slide, 2) education, 3) VM proficiency tests / board exams, and 4) telepathology. Shortcomings include 1) expensive initial setup, 2) inability to maintain an adequate focus in a thick smear with multiple levels, 3) large storage size of the VM slide, and 4) relatively long time needed to scan a slide.