Formation of a functional endothelium on vascular grafts

The lack of a functional endothelial cell lining on artificial polymeric vascular grafts severely reduces their effectiveness in replacing small caliber (< 6 mm) blood vessels. Techniques have now been developed to transplant autologous endothelial cells from one site in the body onto the surface of grafts prior to implantation. Pre-clinical animal trials provide evidence that grafts sodded with autologous, fat-derived, microvessel endothelial cells exhibit a stable, antithrombogenic lining of endothelium. The new endothelial cell lining exhibits morphologies identical with endothelium on native blood vessels. The effectiveness of endothelial cell sodding techniques in pre-clinical animal trials provides support for expanded clinical trials.     

Effects of aging and HIF-1alpha deficiency on permeability of hippocampal vessels

We examined age-related changes in the blood-brain barrier (BBB) of neural cell-specific hypoxia inducible factor-1alpha (HIF-1alpha) deficient mice, which showed hydrocephalus with neuronal cell loss, to investigate an effect of neural cell-specific HIF-1alpha deficiency or hydrocephalus on vascular function. Vascular permeability of horseradish peroxidase (HRP) and binding of cationized ferritin (CF) particles to the endothelial cell luminal surface, as a marker of glycocalyx, were investigated. The thickness of CF-labeled glycocalyx was significantly decreased in the cortex in mutant mice compared with that of control mice, although it was not paralleled by increased vascular permeability. In addition, strong staining for HRP was seen around vessels located along the hippocampal fissure in 24-month-old mutant mice. The reaction product of HRP appeared in an increasing number of the endothelial cell abluminal vesicles and within the thickened basal lamina of arterioles in the hippocampus, showing increased vascular permeability. There were no leaky vessels in 10-week-old mutant mice or 10-week-old and 24-month-old control mice. These findings suggest the necessity of two factors, aging and hydrocephalus, for BBB dysfunction in HIF-1alpha deficient mice.     

In vitro strain-induced endothelial cell dysfunction determined by DNA synthesis

Rapid re-endothelialization following balloon angioplasty can reduce restenosis by inhibiting smooth muscle cell migration and proliferation. However, formation of a neointima following angioplasty can be inhibited due to endothelial cell dysfunction and denudation. The purpose of this study was to evaluate mechanical tensile stress as a cause of endothelial cell dysfunction. The Flexercell strain unit was utilized to generate both short-term cyclic and static tensile strain on cultured bovine aortic endothelial cells (BAECs). Before analysis of this loading on BAECs, strain behaviour of the Flexercell system and DNA assay conditions were optimized. This paper demonstrates that, when compared with unloaded controls, 4-h cyclic loading at 4 per cent elongation and 0.1 Hz, and static loading at 4 per cent elongation cause a 44 and 70 per cent decrease in DNA synthesis respectively. In a companion paper, it is demonstrated that low DNA synthesis levels in mechanically loaded cells can be increased by incubation with Ap4A and/or NO donors.     

Effect of anticoagulation and lavage prior to casting of postmortem material with Mercox® and Batson® 17

In this study we compare the quality of vascular casts, obtained from organs of several animal species from different sources and sacrified under different conditions. Organs from healthy animals were injected with two different polymers such as Mercox and Batson No. 17. When the specimens were observed under a scanning electron microscope structural elements such as endothelial nuclear impressions on vessels and capillaries, endothelial cell borders, venous valves, imprints of smooth muscle cells and intra-arterial cushions were identified. Organs excised post mortem from large animals can be used for microvascular corrosion casting studies with optimal results.     

Histochemical analysis of lymphatic endothelial cells in lymphostasis

The ultrastructure of endothelial cells of intestinal lymphatics and the thoracic duct (TD) and the relation to lymphostasis were examined in rats and monkeys. Localization of 5'-nucleotidase (5'-Nase) and endothelial nitric oxide synthase (eNOS) was studied. In normal lymphatic endothelial cells, 5'-Nase reaction product was evenly deposited on the cell surface in vivo and on cultured TD endothelial cells (TDECs), whereas eNOS was evenly distributed throughout the nucleus and cytoplasm. TDECs had a long filamentous process extending towards the subendothelial extracellular matrix but became flat and regular within 30-40 minutes after gastric perfusion with olive oil. According to their electron-density, two types of cells were found in the TD endothelial layer. The cells with low electron-density exhibited stronger 5'-Nase activity. Valves were bicuspid formations and the valvular endothelial surface of the convex side showed weaker 5'-Nase activity than the concave side. During TD blockage-induced lymphostasis in rats, the 5'-Nase product was almost not discernible in the TDECs within 2 weeks. Larger vesicles were found in the endothelial cytoplasm of the ligated TD. Their number decreased after 6-12 weeks. The small intestinal lymphatics in the mucosa and submucosa were dilated, with numerous open intercellular junctions. The endothelial lining appeared to have reduced activities for 5'-Nase and eNOS in 9 of 11 experimental animals. The results indicated that the inability of the open intercellular junctions, normally working as one-way endothelial flap valves, may be a key morphological feature after TD blockage. Reduced eNOS and 5'-Nase may functionally influence contractile activity and transport capability of the lymphatic vessels in the lymphostasis.     

Angioarchitecture of squamous cell carcinoma from hamster buccal pouch: a scanning electron microscopy study

Tumoral angiogenesis has been widely studied by histochemical analysis but little has been done regarding morphology of these new vessels. The objective of this study was to perform a qualitative analysis of the angiogenic response to chemical induction with dimethylbenzanthracene (DMBA) and carbamide peroxide of squamous cell carcinoma in pouches of Syrian hamsters after different periods of treatment. Twenty‐four Syrian golden hamsters, divided into three groups of eight animals each, had their right jugal pouches treated with a 5% DMBA solution three times a week and a 10% carbamide peroxide two times a week for 55, 70 and 90 days. The left pouch was considered the control. After tumor induction, five animals in each group had their pouches prepared for analysis under scanning electron microscopy and three animals for analysis under light microscopy. The control pouches showed a vascular system composed by few main vessels running parallel to the longest axis of the pouch with some branches. In the pouches submitted to tumor induction, a well‐differentiated squamous cell carcinoma was present since 55 days induction in all samples. The new vascular system showed the presence of many tortuous vessels and the majority of them were veins and capillaries. Terminal loops were extremely sinuous adopting a glomerular or corkscrew shape. These tumor vessels are different from normal vessels, presenting irregular diameters, outpouchings and constrictions. Angiogenesis of sprouting and intussusceptive kind could be identified in the tumor pouches, and they were more frequent as the tumor developed. SCANNING 31: 188–194, 2009. © 2009 Wiley Periodicals, Inc.     

Hormone regulation of endothelial apoptosis and proliferation in vessel regression and angiogenesis

Apoptosis and endothelial proliferation represent two adverse events which take place during vessel regression and angiogenesis, respectively. Apoptosis, an intrinsically activated programmed cell death, regulates cell elimination during vessel regression. In contrast, angiogenesis involves endothelial cell proliferation, migration, and vascular formation. Several molecules, including growth factors and cytokines, produced by endothelial cells and by other cells within the vicinity of the capillary network, regulate apoptosis and angiogenesis. Hormones and endocrine peptides acting via specific receptors located on the endothelial and perivascular stromal cells also have been found to be involved in the regulation of these two major antagonistic processes. The need for a better understanding of the mechanisms involved in hormone regulation of endothelial cell during apoptosis and angiogenesis is of great importance. The accumulating knowledge of hormone regulation may contribute to the introduction of new therapeutic strategies targeting the endothelial cells.     

Expression of VEGFR-3 and 5'-nase in regenerating lymphatic vessels of the cutaneous wound healing

The vascular endothelial growth factor-C (VEGF-C), a specific lymphangiogenic growth factor, raises new questions and perspectives in studying lymphatic development and regeneration. Wound healing skins in mice were processed for 5'-nucleotidase (5'-Nase) and VEGFR-3 (the receptor of VEGF-C) histochemical staining to distinguish lymphatics from blood capillaries and to analyze lymphangiogenesis. In the wounds of 3-5 days after injury, anti-VEGFR-3 immunopositive signals unevenly appeared in 5'-Nase-positive lymphatic vessels in the subcutaneous tissue. A few small circular and irregular lymphatic-like structures with VEGFR-3 expression scattered in the dermal and subcutaneous tissues. Between days 7 and 15 of the wounds, numerous accumulated vasculatures were stained for 5'-Nase and PECAM-1, extending irregularly along the wound edge. Von Willebrand factor was expressed in the endothelial cells of blood vessels and lymphatics in the subcutaneous tissue. Ultrastructural changes of lymphatic vessels developed at different stages, from lymphatic-like structures to newly formed lymphatic vessels with an extremely thin and indented wall. Endothelial cells of the lymphatic vessel were eventually featured by typical intercellular junctions, which deposited with reaction products of VEGFR-3 and 5'-Nase-cerium but lacked VEGF-C expression. The present findings indicate that VEGF-C-induced lymphangiogenesis occurs from the subcutaneous to the dermis along the wound healing edge, especially in the dermal-subcutaneous transitional area, favorable to growth of regenerating lymphatic vessels.     

Mechano-Sensing and shear stress-shielding by endothelial primary cilia: structure,composition, and function

Primary cilium is an antenna-like and non-motile structure protruding from the apical surface of most mammalian cells including endothelial cells lining the inner side of all the blood vessels in our body. Although it has been over a century since primary cilia were discovered, the investigation about their mechano-sensing and other roles in maintaining normal functions of cardiovascular system has just started in recent years. This focused review aims to give an update about the current literature for the role of endothelial primary cilia in blood flow mechano-sensing and shear stress-shielding. To do this, we first summarized the characteristic features of endothelial primary cilia in terms of structure, dimension, molecular composition, and mechanical properties (e.g., bending rigidity), which are the dominant factors for their functions in mechano-sensing and transduction, as well as vascular protection from the blood flow-induced wall shear stress. We also described the experimental techniques and mathematical models for determining the dimension and mechanical properties of the primary cilium. Then we reviewed the molecular mechanisms underlying mechano-sensing and transduction by endothelial primary cilia and the mathematical model prediction for their roles in redistribution and reduction of wall shear stresses. Finally, we briefly discussed the common cardiovascular diseases, e.g., atherosclerosis, hypertension, and aneurysm, due to defects and malfunction of endothelial primary cilia and suggested potential targets for therapeutic treatments.     

Role of adrenocorticotropic hormone in the development and maintenance of the adrenal cortical vasculature

The adrenal cortex is a highly vascularized endocrine tissue. A dense network of blood capillaries centripetally irrigates the adrenal gland, allowing every endocrine cell to be in contact with an endothelial cell. The pituitary hormone ACTH controls the coordinated development of the vasculature and the endocrine tissue mass. This suggests that paracrine secretions between steroidogenic adrenocytes and capillary endothelial cells participate in the control of adrenocortical homeostasis. Besides its effect on the vascular tone of arteries, ACTH induces the expression of the angiogenic cytokine VEGF-A (vascular endothelial growth factor-A) in primary cultures of adrenocortical cells. This growth factor is a specific mitogen for endothelial cells and is likely to mediate the hormonal control of adrenocortical vascularization through a paracrine mechanism. The newly discovered angiogenic factor EG-VEGF (endocrine-gland-derived vascular endothelial growth factor), the expression of which is restricted to endocrine glands and which is preferentially mitogenic for endocrine tissue-derived endothelial cells, is another candidate mediator of great potential interest.     

Interaction of rat tumor cells with blood vessels and lymphatics of the avian chorioallantoic membrane

It has generally been assumed that tumors do not induce lymphangiogenesis and only very recently animal models have been presented showing tumor-induced lymphangiogenesis. We have grown two types of rat tumor cells, 10AS pancreatic carcinoma and C6 glioma cells, on the chorioallantoic membrane (CAM) of chick and quail embryos. The suspended tumor cells rapidly formed solid tumors which invaded the CAM and were vascularized by CAM vessels. When grown on the CAM of quail embryos intratumoral endothelial cells could be specifically stained with the QH1 antibody. In C6 gliomas the vascular pattern was more regular than in 10AS carcinomas. The vessels often grew radially into the glioma and many of them were invested by smooth muscle alpha-actin-positive periendothelial cells. Lymphatics, which were identified by vascular endothelial growth factor receptor-3 (VEGFR-3) in situ hybridization were absent from C6 gliomas, although a weak expression of the lymphangiogenic growth factor, VEGF-C, could be detected in the C6 cells by Northern blot analysis. In contrast, 10AS cells, which expressed high levels of VEGF-C, induced ingrowth of lymphatics into the tumors, with BrdU-labeling rates of about 9% of lymphatic endothelial cells. Our studies demonstrate the heterogeneity of interactions of tumor cells with blood vessels and lymphatics and show that sufficient quantities and/or quality of lymphangiogenic growth factors are crucial for the induction of lymphatics in tumors.     

Embryonic erythropoiesis in human yolk sac: two different compartments for two different processes

The wall of 12 yolk sacs (YSs) from 17- to 50-day-old human embryos was examined by light, scanning, and transmission electron microscopy to identify the ontogeny of embryonic erythropoiesis. Initial formation of blood island with the generation of erythroid and endothelial cells was seen in the mesenchymal layer in embryos aged 17 days. A network of blood vessels containing abundant erythroblasts was identified in the YS walls of embryos aged approximately 24 days. At this age, erythroblasts were also identified within the embryo body. Primitive erythroblasts were the only cells present within the embryo and its YS until the end of week 5. These cells first appeared in the mesenchymal vascular plexus of the YS wall, and were then observed in the liver and other tissues of the embryo. At embryonic week 5, two compartments were identified in the YS wall; a mesodermal one in which blood vessels were formed, and an endodermal compartment in which erythrocytes were present within the endodermal vesicles. Erythrocytes were small non-nucleated cells similar to adult erythrocytes. Transmission electron microscopic observation focused on the endodermal vesicles confirmed the presence of definitive erythrocytes only at such extra vascular location. At this age, there were no definitive erythrocytes detected within the embryo. Erythrocytes started to be identified in embryonic blood vessels from week 7 onward. These findings provide information not previously described about YS erythropoiesis during early human development.     

Ultrastructure of endothelial cells under flow alteration

Endothelial cells are stable and quiet in normal animals. They arrange regularly and have a smooth lumen surface and thin endothelial wall. According to Thoma's principle (1893) and Kamiya and Togawa's principle (1980) on the relationship of the vascular diameter to flow alteration, blood flow is in equilibrium to the diameter and in a physiological state. That is to say, there is no fast flow or slow flow. To understand the nature of the endothelial cells, we should investigate endothelial cells under flow alteration to break the equilibrium state. Endothelial cells under increased flow were studied in arteries with an arteriovenous fistula or in the capillaries of myocardium with volume-overloaded hearts or of the skeletal muscle by electrical stimulation. Those under decreased flow were studied by the closure of the fistula or by ceasing the stimulation. Endothelial cells in the coarctation of the arteries were also observed. Endothelial cells were activated by increased flow in the arteries and capillaries, while they were inactivated by decreased flow. Endothelial activation is characterized as lumen protrusions, increase of cytoplasmic organelles, abluminal protrusions, basement membrane degradation, internal elastic lamina degradation in the arteries, and sproutings in the capillaries. These are ultrastructurally comparable to angiogenesis. Endothelial inactivation is characterized by the decrease of endothelial cell number with apoptosis, which is ultrastructurally comparable to angioregression. We assume that endothelial cells respond to increased flow by angiogenesis and to decreased flow by angioregression.     

Quantitative analysis of the number of antigens immobilized on a glass surface by AFM

To develop force measurements using an atomic force microscope (AFM) in a quantitative manner, it is necessary to estimate the number density of target molecules on a sample surface, and for this, the sensitivity of detection should be known. In this study, the AFM was used as a mechanical detector and an antigen and its antibody were used as a model to evaluate the sensitivity of detection. Antigens were immobilized on a glass surface and number density was estimated by monitoring optical absorbance due to product formation by the reaction of crosslinkers. The concentration of antigen was controlled by mixing control peptides. A microbead was used as a probe and antibodies were immobilized on the bead. AFM force measurements were then made for a range of number densities in the order of 10–10⁶ antigen molecules per square micrometer of surface and were compared to evaluate the sensitivity of detection. Our result establishes the reliability of estimating a number of molecules like receptors on the cell surface, and indicates that the AFM is useful as a mechanical detector with high sensitivity.     

Tissue expression of platelet endothelial cell adhesion molecule-1 at pre and postnatal murine development.

Endothelial cells, at the cell-cell borders, express PECAM-1, and have been implicated in vascular functions. The monoclonal antibody MEC 13.3 recognizes PECAM-1 molecule from mouse vessels and allows to analyze the ontogeny of mouse endothelium. At the present, little is known about the molecular basis of differentiation pathways of endothelial cells, that enables its morphological heterogeneity. The purpose of this study was to analyze the pattern of PECAM-1 expression, employing monoclonal antibody MEC 13.3, in cellular suspensions obtained from different mouse organs at pre and postnatal stages. Fluorescence activated cell sorter analysis showed a different profile of the glycoprotein expression in a cell population with size and granularity selected by 1G11 endothelial cell line. The expression differs from prenatal to postnatal developmental stages in a given organ, and among the organs studied. Another cell population, with a size and granularity higher than IG11 endothelial cell line, coexists in cellular suspensions obtained from liver, gut and brain. These cells could be related to those detected by means of immunoenzyme methods which showed a non-differentiated morphology. The different PECAM-1 pattern expression could reflect potential organ-specific differentiation pathways during development and according to organs environment. The existence of another cell population with a size and granularity higher than 1G11 endothelial cell line required a phenotypic characterization.     

Analysis of elastohydrodynamic lubrication in McKee-Farrar metal-on-metal hip joint replacement

An elastohydrodynamic lubrication (EHL) analysis was carried out in this study for a typical McKee-Farrar metal-on-metal hip prosthesis under a simple steady state rotation. The finite element method was used initially to investigate the effect of the cement and bone on the predicted contact pressure distribution between the two articulating surfaces under dry conditions, and subsequently to determine the elastic deformation of both the femoral and the acetabular components required for the lubrication analysis. Both Reynolds equation and the elasticity equation were coupled and solved numerically using the finite difference method. Important features in reducing contact stresses and promoting fluid-film lubrication associated with the McKee-Farrar metal-on-metal hip implant were identified as the large femoral head and the thin acetabular cup. For the typical McKee-Farrar metal-on-metal hip prosthesis considered under typical walking conditions, an increase in the femoral head radius from 14 to 17.4 mm (for a fixed radial clearance of 79 microm) was shown to result in a 25 per cent decrease in the maximum dry contact pressure and a 60 per cent increase in the predicted minimum film thickness. Furthermore, the predicted maximum contact pressure considering both the cement and the bone was found to be decreased by about 80 per cent, while the minimum film thickness was predicted to be increased by 50 per cent. Despite a significant increase in the predicted minimum lubricating film thickness due to the large femoral head and the thin acetabular cup, a mixed lubrication regime was predicted for the McKee-Farrar metal-on-metal hip implant under estimated in vivo steady state walking conditions, depending on the surface roughness of the bearing surfaces. This clearly demonstrated the important influences of the material, design and manufacturing parameters on the tribological performance of these hard-on-hard hip prostheses. Furthermore, in the present contact mechanics analysis, the significant increase in the elasticity due to the relatively thin acetabular cup was not found to cause equatorial contact and gripping of the ball.     

Diversity in unity: the biochemical composition of the endothelial cell surface varies between the vascular beds

The vascular endothelium represents a population of squamous epithelial cells characterized by a particular histological localization (intima of blood vessels) and by several physiological functions such as the transport of substances between blood and tissues, the modulation of the vascular tone, the control of blood coagulation and that of the leukocyte extravasation. In spite of all these elements in common and of an identical embryonic origin, endothelial cells show definite morphological and physiological variations that divide them into types and subtypes, each specifically associated to various categories of organs. Even within the vasculature of the same organ, there are clear segmental (arterial/capillary/venous) differentiations of the endothelial cells. While the morphological and physiological differences between endothelial cells are well documented, there are very few data on the biochemistry underlying this heterogeneity. This work presents several data suggesting that, at present, the domain is ripe for a comprehensive analysis of this biochemical diversity, at least in what concerns the luminal aspect of the endothelial plasmalemma, a compartment of crucial importance in the biology and pathology of the cardiovascular system.     

大变形量下高碳钢环件冷轧变形过程模拟与试验研究

研究了大变形量下高碳钢环件冷轧变形行为,利用有限元模拟方法分析了环件冷轧过程中的形变规律,采用光学显微镜、SEM等材料表征手段研究了大变形量下环件冷轧过程中的组织演化特点。结果表明：在较小的变形量下,环件内外侧应变较中间应变大,且变形首先发生在环件外侧;整个环件变形过程中,环件中间层的晶粒变形程度最小,外层次之,内层的变形最为剧烈;铁素体基体沿轧制方向呈现明显的方向性,碳化物颗粒分布更为均匀,且数量变少;随着变形量的增大,环件内层的应变明显大于外层的应变,且最小应变的位置偏移至靠外层比较近的区域,其组织中碳化物颗粒脱落加重;当变形量达到625%时,环件达到塑性极限,在内侧表面产生裂纹发生破坏。     

Expression of adhesion molecules in the lower uterine segment during term and preterm parturition

Term and preterm cervical ripening and dilatation have similarities with an inflammatory reaction. Since cell adhesion molecules are involved in this process, investigations on the expression of intercellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, vascular cell adhesion molecule-1, and platelet endothelial cell adhesion molecule in the lower uterine segment and in vitro experiments on human umbilical vein endothelial cells were performed. In addition, current reports on expression of endothelial adhesion molecules by the uterine cervix were summarized. Cell adhesion molecule expression by lower uterine segment and uterine cervix in term and preterm parturition was measured using immunohistochemistry, enzyme immunoassay, and Northern blot analysis. Regulation of adhesion molecule expression was evaluated in vitro by indirect immunofluorescence and flow cytometry using human umbilical vein endothelial cells. Investigations in term parturition revealed that intercellular adhesion molecule-1, endothelial leukocyte adhesion molecule-1, and vascular cell adhesion molecule-1 expression increases during parturition. In preterm labor, the expression of endothelial leukocyte adhesion molecule-1 and intercellular adhesion molecule-1 in the lower uterine segment increased. Expression of platelet endothelial cell adhesion molecule did not change in term and preterm parturition. Expression of adhesion molecules was localized mainly on lower uterine segment vascular endothelial cells and to a smaller extent on leukocytes. In vitro experiments showed that expression of adhesion molecules by human umbilical vein endothelial cells can be stimulated by tumor necrosis factor-alpha, 17beta-estradiol, prostaglandin E(2), and the antigestagen onapristone. Progesterone exerted no stimulatory effect. Cervical ripening and dilatation during term and preterm parturition are associated with an increased expression of endothelial cell adhesion molecules by lower uterine segment and uterine cervix. The expression can be modulated by pro-inflammatory cytokines, sex hormones, and prostaglandin E(2). Mechanisms controlling the extravasation of leukocytes may play a fundamental role in term and preterm parturition.