Transcytosis of plasma macromolecules in endothelial cells: a cell biological survey

The modern exploration of endothelial cell biology is a largely interdisciplinary exercise. Cell biological, physiological, and more recently molecular biology approaches were used to study the pathways and the organelles involved in transcytosis of macromolecules in endothelial cell (EC). Here we discuss mainly the cell biological findings that revealed that EC have the attributes to fulfill the transport function. They are polarized cells, heterogeneous, and, thus, structurally and functionally adapted to the vascular bed in which they reside. The structural heterogeneity involves the number and distribution of plasmalemmal vesicles (caveolae), their generated channels, and the organization of intercellular junctions. The closely related functional heterogeneity comprises the degree of permeability for plasma molecules that vary as a function of organ. The EC are endowed with the cellular machinery to perform (1) endocytosis, that is to take up plasma proteins and the molecules they carry to be used for themselves (cholesterol-carrying low density lipoproteins, fatty acid carrying albumin, iron carrying transferrin, etc.), and (2) transcytosis, which implies to transport plasma proteins to the subjacent cells and tissues. The possible pathways for transport of molecules are transcellular, via caveolae and channels, and paracellular via intercellular junctions. Most of the results obtained, so far, indicate that transcytosis of albumin, low-density lipoproteins, metaloproteases, and insulin, is performed by cargo-vesicles and their generated channels. The paracellular pathway can be used for water and ions; in postcapillary venules, at the level of which approximately 30% of junctions are open to a space of 6 nm, small molecules may take this route. Recent data obtained by molecular biology techniques revealed that caveolae are endowed with the molecular machinery for fusion/fission, docking, and movement across cells. Moreover, the various and numerous molecules that have been detected in the caveolae membrane and the different functions assumed by this differentiated microdomain strengthen the postulate that there are at least two or more types of vesicles molecularly tailored for the local physiological requirements.     

Effects of rotational culture on morphology,nitric oxide production and cell cycle of endothelial cells

Devices for the rotational culture of cells and the study of biological reactions have been widely applied in tissue engineering. However, there are few reports exploring the effects of rotational culture on cell morphology, nitric oxide (NO) production, and cell cycle of the endothelial cells from human umbilical vein on the stent surface. This study focuses on these parameters after the cells are seeded on the stents. Results showed that covering of stents by endothelial cells was improved by rotational culture. NO production decreased within 24 h in both rotational and static culture groups. In addition, rotational culture significantly increased NO production by 37.9% at 36 h and 28.9% at 48 h compared with static culture. Flow cytometry showed that the cell cycle was not obviously influenced by rotational culture. Results indicate that rotational culture may be helpful for preparation of cell-seeded vascular grafts and intravascular stents, which are expected to be the most frequently implanted materials in the future.     

同科室两种生化检测系统对10项生化项目测定结果的比对和偏倚分析

目的:通过本科室两种生化检测系统(BDS)对10项常规生化项目的检验结果进行比对分析和偏倚评估,探讨不同BDS对同一检测项目的结果是否具有可比性和偏倚可接受性。方法:根据美国临床实验室标准协会EP9-A2文件标准要求,每天随机选取8例新鲜血清样本在贝克曼AU2700[为参比系统]和XC8001[为试验系统]全自动生化分析仪上测定10项(谷丙转氨酶、谷草转氨酶、白蛋白、总蛋白、肌酐、尿素、总胆固醇、甘油三酯、高密度脂蛋白胆固醇和低密度脂蛋白胆固醇)常规生化项目,每例样本按正反序重复检测两次,连续测量5天,记录各测定值。计算Y与X之间的相关系数,直线回归方程和不同医学决定水平处的相对偏差。并以美国临床实验室改进修正案能力验证规则(CLIA,88)分析质量要求中规定可接受范围的1/2作为标准,判断两BDS之间的可比性和偏倚临床可接受性。结果:未发现有离群点出现,10个常规生化检测项目的r值均≥0.975,满足EP9-A2文件规定r≥0.975(或R2≥0.95)的要求。所检测数据经单因素方差分析,差异无统计学意义(P>0.05)。10个常规生化检测项目不同Xc的相对偏差SE%,结果均<可接受范围的1/2。结论:本院两种BDS通过方法学比对,对同一检测项目测定结果具有一致性,且各检测项目在各医学决定水平处出现偏倚临床是接受可接受性。     

Morphologic and temporal analysis of vascular smooth muscle cell apoptosis induced by c-Myc and E1A

Apoptosis is a physiologic form of cell death present in many disease conditions. When the balance of mitosis versus apoptosis is altered, tumor-like growth or degeneration of tissues may ensue. This appears to occur in several diseases, including those of the cardiovascular system, where apoptosis plays a key role in atherosclerosis and restenosis following angioplasty. Since c-myc is upregulated in the pathogenesis of these diseases, we chose to study the sequential morphologic features of programmed cell death in vascular smooth muscle cells induced by c-myc and by the adenovirus early gene E1A. Morphology and timed events in apoptotic cell cultures were analyzed by scanning electron microscopy, transmission electron microscopy, and time-lapse videomicroscopy. We observed that both c-myc-and E1A-induced apoptosis (in serum-free medium) resulted in numerous, tightly packed clusters of apoptotic blebs, as well as in one or two asymmetrically larger blebs. Transmission electron microscopy analysis revealed the larger blebs contained mostly nuclear chromatin, whereas the many smaller fragments often had little or no chromatin. Time-lapse studies showed that apoptosis was induced at a slower rate in cells stably transfected with c-myc versus those stably transfected with E1A. The early changes of apoptosis, including cell shrinkage and intense blebbing, occurred in under 5 min in both cells. Slight alterations such as cell size and further rounding occurred up to 8 h following the initial changes of apoptosis. Rather than being a part of the apoptotic response, release from the culture floor almost entirely resulted from movement of the culture flask. These studies provide a framework of timed morphologic events for future mechanistic investigation into the key aspects of myc-and E1A-induced apoptosis in vascular smooth muscle.     

乳液润滑剂表面张力与组分相关性研究

表面张力是乳化液的基本性能之一,它对乳液润滑剂的润湿性及离水展着能力有重要影响。本文考察了基础油及不同类型极性添加剂等因素对乳化液表面张力的影响,研究表明：乳液组分若能有效提高气一液界面层分子结合的紧密程度,将有利于降低乳液的表面张力。     

Histological demonstration of increased vascular permeability in the dura mater of the rat

Neurogenic inflammation of the dura mater encephali has been suggested to play an important role in the pathophysiology of headaches. Although functional studies using extravasation techniques indicate an enhanced permeability of blood vessels after chemical or electrical stimulation of C-fibres supplying the dura mater, histological demonstration of leaky blood vessels is still a problem. We used the vascular labelling method combined with i.v. injection of colloidal silver solution to test the permeability increasing effect of intravenous administration of substance P, topical application of mustard oil or acidic phosphate buffer and local electrical stimulation of the exposed dura mater. Histological characteristics of increased vascular permeability were observed exclusively after mustard oil and acidic phosphate buffer. This observation may indicate different mechanisms of increased vascular permeability involving pinocytosis and formation of interendothelial gaps selectively visualized by the vascular labelling method.     

Angiofil-mediated visualization of the vascular system by microcomputed tomography: a feasibility study

Visualization of the vascular systems of organs or of small animals is important for an assessment of basic physiological conditions, especially in studies that involve genetically manipulated mice. For a detailed morphological analysis of the vascular tree, it is necessary to demonstrate the system in its entirety. In this study, we present a new lipophilic contrast agent, Angiofil, for performing postmortem microangiography by using microcomputed tomography. The new contrast agent was tested in 10 wild-type mice. Imaging of the vascular system revealed vessels down to the caliber of capillaries, and the digital three-dimensional data obtained from the scans allowed for virtual cutting, amplification, and scaling without destroying the sample. By use of computer software, parameters such as vessel length and caliber could be quantified and remapped by color coding onto the surface of the vascular system. The liquid Angiofil is easy to handle and highly radio-opaque. Because of its lipophilic abilities, it is retained intravascularly, hence it facilitates virtual vessel segmentation, and yields an enduring signal which is advantageous during repetitive investigations, or if samples need to be transported from the site of preparation to the place of actual analysis, respectively. These characteristics make Angiofil a promising novel contrast agent; when combined with microcomputed tomography, it has the potential to turn into a powerful method for rapid vascular phenotyping.     

Time‐series investigation of fused vesicles in microvessel endothelial cells with atomic force microscopy

Vesicles or caveolae within endothelial cells, fusing together to form vacuolar organelles, are implicated in macromolecular transport and cellular element transmigration across the blood–brain barrier (BBB) during inflammation and ischemia. Vacuolar organelles have been described by transmission electron microscopy and immunofluorescence, but the details of their dynamics have not been well addressed yet. Herein, by using tapping mode atomic force microscopy (AFM), we observed the time‐series changes of fused vesicles within the serum‐free cultured rat cerebral microvessel endothelial cells. The fused vesicles were certainly proved by fluorescent staining of Fm4‐64 combining simultaneous AFM imaging, as well as the field emission scanning electron microscopy technique. And energy dispersive spectrum results additionally implied that there may be specific structure and compositions around the vesicle region. These results indicate that increased vesicles in BBB may contribute to the formation of fused vesicles and a higher probability to construct the trans‐endothelial channel across endothelium layer. Furthermore, the AFM application may open up a new approach to investigate the details of transcellular process by fused vesicles. Microsc. Res. Tech., 2010. © 2009 Wiley‐Liss, Inc.     

Cytoskeletal response of microvessel endothelial cells to an applied stress force at the submicrometer scale studied by atomic force microscopy

Cytoskeleton fibers form an intricate three-dimensional network to provide structure and function to microvessel endothelial cells. During accommodation to blood flowing, stress fiber bundles become more prominent and align with the direction of blood flow. This network either mechanically resists the applied shear stress (lateral force) or, if deformed, is dynamically remodeled back to a preferred architecture. However, the detailed response of these stress fiber bundles to applied lateral force at submicrometer scales are as yet poorly understood. In our in vitro study, the tip, topography probe in lateral force microscopy of atomic force microscopy, acted as a tool for exerting quantitative vertical and lateral force on the filaments of the cytoskeleton. Moreover, the authors developed a formula to calculate the value of lateral force exerted on every point of the filaments. The results show that cytoskeleton fibers of healthy tight junctions in rat cerebral microvessel endothelial cells formed a cross-type network, and were reinforced and elongated in the direction of scanning under lateral force of 15-42 nN. Under peroxidation (H(2)O(2) of 300 micromol/L), the cytoskeleton remodeled at intercellular junctions, and changed over the meshwork structures into a dense bundle, that redistributed the stress. Once mechanical forces were exerted on an area, the cells shrank and lost morphologic tight junctions. It would be useful in our understanding of certain pathological processes, such as cerebral ischemia/reperfusion injury, which maybe caused by biomechanical forces and which are overlooked in current disease models.     

Lymphatic vessels in the oral cavity: different structures for the same function.

A study using a light and transmission electron microscope was performed on some structural characteristics of the lymphatic capillaries in different regions of the human oral cavity. The lymphatic capillaries of dental pulp, masticatory mucosa (gingiva and peri-implant mucosa) and lining mucosa (cheek) were examined. Our attention was focused on the morphologic characteristics of the endothelial wall in the lymphatic capillaries. In particular, the connections between endothelial cells were investigated. In the lymphatic capillaries of the dental pulp, the endothelial wall was always very complex. It frequently presented protrusions of the endothelial cells that overlapped and formed intercellular channels. These channels were thus contained by the vessel endothelial wall with their extremities opening out towards the surrounding interstitium and the vessel lumen. The endothelial wall of the lymphatic capillaries of the cheek was very smooth and thin without complex intercellular junctions. The endothelial cells were joined by end-to-end junctions and open junctions were frequently observed. Intercellular channels were also found in the endothelial wall of lymphatic capillaries of the gingiva and the peri-implant mucosa. The presence of numerous clefts represented by the open junctions in the lymphatics of the cheek and the existence of complex intercellular adhesions with the formation of intercellular channels in the endothelial wall of the lymphatic capillaries of the dental pulp and gingiva induce us to believe that these may play a role in the various mechanisms used by lymphatic capillaries to absorb interstitial fluids. These mechanisms are based on the different morpho-functional characteristics of the surrounding tissue.     

Comparative atomic force and scanning electron microscopy: an investigation on fenestrated endothelial cells in vitro

Rat liver sinusoidal endothelial cells (LEC) contain fenestrae, which are clustered in sieve plates. Fenestrae control the exchange of fluids, solutes and particles between the sinusoidal blood and the space of Disse, which at its back side is flanked by the microvillous surface of the parenchymal cells. The surface of LEC can optimally be imaged by scanning electron microscopy (SEM), and SEM images can be used to study dynamic changes in fenestrae by comparing fixed specimens subjected to different experimental conditions. Unfortunately, the SEM allows only investigation of fixed, dried and coated specimens. Recently, the use of atomic force microscopy (AFM) was introduced for analysing the cell surface, independent of complicated preparation techniques. We used the AFM for the investigation of cultured LEC surfaces and the study of morphological changes of fenestrae. SEM served as a conventional reference.
AFM images of LEC show structures that correlate well with SEM images. Dried-coated, dried-uncoated and wet-fixed LEC show a central bulging nucleus and flat fenestrated cellular processes. It was also possible to obtain height information which is not available in SEM. After treatment with ethanol or serotonin the diameters of fenestrae increased (+6%) and decreased (−15%), respectively. The same alterations of fenestrae could be distinguished by measuring AFM images of dried-coated, dried-uncoated and wet-fixed LEC. Comparison of dried-coated (SEM) and wet-fixed (AFM) fenestrae indicated a mean shrinkage of 20% in SEM preparations. In conclusion, high-resolution imaging with AFM of the cell surface of cultured LEC can be performed on dried-coated, dried-uncoated and wet-fixed LEC, which was hitherto only possible with fixed, dried and coated preparations in SEM and transmission electron microscopy (TEM).     

The roles of focal adhesion and cytoskeleton systems in fluid shear stress-induced endothelial cell response

Focal adhesions are polyproteins linked to extracellular matrix and cytoskeleton, which play an important rolein the process of transforming force signals into intracellular chemical signals and subsequently triggering relatedphysiological or pathological reactions. The cytoskeleton is a network of protein fibers in the cytoplasm, which iscomposed of microfilaments, microtubules, intermediate filaments, and cross-linked proteins. It is a very importantstructure for cells to maintain their basic morphology. This review summarizes the process of fluid shear stresstransduction mediated by focal adhesion and the key role of the cytoskeleton in this process, which focuses on thefocal adhesion and cytoskeleton systems. The important proteins involved in signal transduction in focal adhesion areintroduced emphatically. The relationship between focal adhesion and mechanical transduction pathways arediscussed. In this review, we discuss the relationship between fluid shear stress and associated diseases such asatherosclerosis, as well as its role in clinical research and drug development.     

Microvascular growth, development, and remodeling in the embryonic avian kidney: the interplay between sprouting and intussusceptive angiogenic mechanisms

Embryonic development is associated with extensive vascular growth and remodeling. We used immunohistochemical, light and electron microscopical techniques, as well as vascular casting methods to study the developing chick embryo kidney with special attention to the interplay between sprouting and intussusceptive vascular growth modes. During inauguration at embryonic day 5 (E5), the early mesonephros was characterised by extensive microvascular sprouting. By E7, the vascular growth mode switched to intussusception, which contributed to rapid kidney vasculature growth up to E11, when the first obvious signs of vascular degeneration were evident. The metanephros underwent similar phases of vascular development inaugurating at E8 with numerous capillary sprouts and changing at E13 to intussusceptive growth, which was responsible for vascular amplification and remodeling. A phenomenal finding was that future renal lobules arose as large glomerular tufts, supplied by large vessels, which were split into smaller intralobular feeding and draining vessels with subsequent formation of solitary glomeruli. This glomerular duplication was achieved by intussusception, i.e., by formation of pillars in rows and their successive merging to delineate the vascular entities. Ultimately, the maturation of the vasculature was achieved by intussusceptive pruning and branching remodeling. An interesting finding was that strong VEGF expression was associated with the sprouting phase of angiogenesis while bFGF was upregulated during the phase of intussusceptive microvascular growth. We conclude that microvascular growth and remodeling in avian kidney follows an adroitly crafted pattern, which entails a precise spaciotemporal interplay between sprouting and intussusceptive angiogenic growth modes supported partly by VEGF and bFGF.     

Angiogenesis in endocrine glands: special reference to the expression of vascular endothelial growth factor

Recent studies have shown that several angiogenic growth factors are produced and secreted by normal endocrine cells and are increased in pathological states of endocrine glands, including inflammation, hyperplasia, and neoplasia. Expression of corresponding receptors on epithelial cells and/or endothelial cells enables these angiogenic factors to influence growth and function of the endocrine tissues by auto- or paracrine mechanisms. Some of the angiogenic factors are also considered to be involved in angiogenesis, which is a critical process in tumor formation and progression. Vascular endothelial growth factor (VEGF) is regarded as one of most important angiogenic factors with specific effects on endothelial cell growth and vascular permeability, and is isolated from a variety of normal and neoplastic endocrine cells. In this article, recent studies on angiogenic factors, especially on expression of VEGF, are reviewed in the field of endocrine systems.     

Microvasculature in gingivitis and chronic periodontitis: disruption of vascular networks with protracted inflammation.

Gingivitis occurring when bacterial plaque accumulates in the gingival crevice provides a convenient and interesting model for chronic inflammation in humans. In some patients, gingivitis progresses to the destructive lesion of periodontitis, involving the formation of periodontal pockets. The basis for pocket formation and progression is not as yet clear, although neutrophilic polymorphonuclear leukocytes (PMN) appear to play a protective role. Vascular changes appear to either facilitate or inhibit PMN function with the effect of either protecting from, or stimulating, periodontitis. Contrary to most circumstances, high endothelial cells in periodontitis are involved with PMN rather than lymphocyte emigration. Expansion of the microvasculature through increased vascular diameter and tortuosity as well as the development of high endothelial cells appears to protect from periodontitis by increasing the supply of both plasma defense factors and PMN to the tissues. Vascular changes that may oppose this and promote periodontitis are the formation of perivascular hyaline material and accumulation of basement membrane rests. The inadequate tissue turnover that accumulation of these vascular products represents can be argued as a vascular response to a chronic inflammation that has failed to eliminate the irritant. It is suggested that these vascular changes may account for the highly localized and burst-like pattern of pocket formation in periodontitis. Finally, it is possible that the recent observation that periodontitis is an independent risk factor for systemic vascular disease may reflect stimulation of acute phase protein synthesis by cytokines released by periodontal high endothelial cells.     

Angioarchitecture of the terminal vascular system in inflammatory bowel disease—A comparative corrosion-anatomic study

The vascular origin of Crohn's disease (CD) and ulcerative colitis has been discussed but is poorly understood and investigated. A comparison of both diseases on the basis of the angioarchitecture had yet to be performed to investigate vascular morphology further and to evaluate its relevance for the etiology and differential diagnosis. The vascular system of 29 human colonic specimens of patients with CD (n=10), ulcerative colitis (n=11), and colon cancer (n=8), which were taken as controls, was perfused blood-free immediately after resection. Corrosion casts were obtained by injection of Mercox Cl and consecutive digestion with potassium hydroxide. Casts were then cut into sections, sputter coated with gold, and examined in a blinded fashion by scanning electron microscopy. Normal intestine showed a typical honeycomb appearance, the mucosal capillary network was smooth and regular, and the subepithelial vascular plexus was supplied by regularly arranged arterioles. In CD, a partially intact capillary net with alterations, dominant collecting veins, and a characteristic pattern of diameter changes with narrow segments in the capillary bed was seen. In ulcerative colitis a disruption of the colonic vascular architecture was found. The capillarization was rarefied and irregular lumps of subepithelial capillary structures were observed. The morphology of the terminal vascular bed in inflammatory bowel disease in contrast to normal controls is uniquely described. The technique of injecting fresh, human specimens was applied for the first time. The homogeneity of vascular changes within each entity seems to allow a differentiation between CD and ulcerative colitis.     

Characterization of the primo vascular system in rabbit vagina

The primo vascular system (PVS) is observed in different parts of the body under different physiological and disease conditions. Previously, the PVS was not observed in the vagina. The vaginal samples of this study were collected from the female genitalia of healthy New Zealand white rabbits from the animal house, Faculty of Medicine, Assiut University. The vaginal samples were fixed in Bouin's solution. The sections were stained with hematoxylin and eosin and Crossmon's trichrome. Additionally, the sections were immunohistochemically stained with neuron-specific enolase (NSE) and vascular endothelial growth factor (VEGF). A primo node was observed on the lymph vessel of the vagina and has several characteristics that resemble those of the previously discovered primo nodes. The primo node in this study was surrounded by mesothelial cells that provide positive immunoreactivity to NSE and VEGF. Sinuses of different sizes, floating cells, telocyte-like cell, and primo microcells were observed as the main constituents of the primo node. Additionally, migratory cells were detected, which passed from the primo node to the enclosing lymph vessel.