Ultrastructure of coronary microvessels in conditions of heart reperfusion following prolonged ischemia while applying various methods of artificial hypothermia

It has been found out that in children with Roger's disease corrected in the conditions of two fundamentally different procedures of anesthetic management, myocardial reperfusion after cardiac arrest under artificial hypothermic circulation is accompanied by obstruction of more than 30% of coronary bed microvessels with hydropic endothelial cells or their cystiform fragments. The content of necrotic cells increases, while the "working" cells demonstrate a decrease in myocropinocytotic transport characteristics. Circulatory arrest under perfusionless hypothermia and immersion reperfusion do not result in a dramatic change of general morphology of microvessels as compared to the control group, while a heterogenic response of the structures responsible for transendothelial transfer of macromolecules provides the basis for recovery of the endothelium structure and function, as a patient's temperature reaches a standard value.     

The role of specific endothelial granules in changes of myocardium microvessel reperfusion at open-heart operations under hypothermia

For patients with congenital heart defects, a comparative analysis was carried out of specific endothelial granules and morphological properties of perfusion capacity of microvessels at various stages of reperfusion after prolonged circulatory arrest. At all stages of reperfusion, the quantity of specific granules in microvessel endothelium dramatically dropped, as compared with the control group. At the early stages of reperfusion, the level of granule exocytosis does not lead to any statistically significant changes in perfusion characteristics in the right atrium microvessels, as compared with the control group. Conversely, at the late stages of reperfusion, degranulation of endothelial cells is enhanced, and physiological mechanisms intended to remove the microvessels from circulation are replaced with pathological ones. These results suggest the existence of a threshold in exocytosis of specific endothelial granules, whose exceeding may switch on some pathological mechanisms in blood flow regulation.     

Brain Iron Toxicity: Differential Responses of Astrocytes,Neurons, and Endothelial Cells

Iron accumulation or iron overload in brain is commonly associated with neurodegenerative disorders such as Parkinson’s and Alzheimer’s diseases, and also plays a role in cellular damage following hemorrhagic stroke and traumatic brain injury. Despite the brain’s highly regulated system for iron utilization and metabolism, these disorders often present following disruptions within iron metabolic pathways. Such dysregulation allows saturation of proteins involved in iron transport and storage, and may cause an increase in free ferrous iron within brain leading to oxidative damage. Not only do astrocytes, neurons, and brain endothelial cells serve unique purposes within the brain, but their individual cell types are equipped with distinct protective mechanisms against iron-induced injury. This review evaluates iron metabolism within the brain under homeostatic and pathological conditions and focuses on the mechanism(s) of brain cellular iron toxicity and differential responses of astrocytes, neurons, and brain vascular endothelial cells to excessive free iron. Special issue dedicated to Dr. Moussa Youdim. An erratum to this article can be found at     

定量研究脑缺血及再灌注下微血管的通透性

利用示踪剂ＦＬＮａ在脑缺血及再灌注的动物模型上,通活体观察和测定血液、脑等脏 荧光强度,以及对软脑膜微血管荧光光图象的平滑处理与定量分析,研究软脑膜微血管的通透性,探讨脑缺血及再灌注对微血管通透性的影响及内在规律。实验结果表明：缺血、缺血及再灌注会引起微血管内皮细胞的损伤,导致微血管通透性增大,这种损伤一般发生在缺血或再灌注早期,早然各脏器微血管都受到损伤,但其荧光值不同,说明各脏器抗缺血与缺氧的     

Cerebral microvessels and derived cells in tissue culture: Isolation and preliminary characterization

Summary Microvessels isolated from mouse forebrain were used as the source material for the derivation of cerebral vascular endothelium and smooth-muscle cells in culture. The microvessels were isolated by a mechanical dispersion and filtration technique, and were maintained in vitro as organoid cultures. A microvessel classification system was developed and proved to be useful as a tool in monitoring culture progress and in predicting the type(s) of microvessel(s) that would give rise to migrating and/or proliferating cells. The isolated cerebral microvessels were heterogeneous in diameter, size of individual vascular isolate, and proliferative potential. The isolated microvessels ranged in diameter from 4 μm to 25 μm and in size from a single microvascular segment to a large multibranched plexus with mural cells. The initial viability, determined by erythrosin B exclusion, was approximately 50% on a per cell basis. All microvessel classes had proliferative potential although the rate and extent of proliferation were both microvessel class- and density-dependent. The smaller microvessels gave rise to endothelial cells, whereas the large microvessels gave rise to endothelial and smooth-muscle cells. The viability and progress of a microvessel toward derived cell proliferation seemed to be directly proportional to the number of mural cells present. This work was supported in part by an Arteriosclerosis Specialized center of Research grant from the National Heart, Lung and Blood Institute, National Institutes of Health (HL-14230) and Grant 584-127703 from the Veterans Administration.     

Effects of curvature and cell–cell interaction on cell adhesion in microvessels

It has been found that both circulating blood cells and tumor cells are more easily adherent to curved microvessels than straight ones. This motivated us to investigate numerically the effect of the curvature of the curved vessel on cell adhesion. In this study, the fluid dynamics was carried out by the lattice Boltzmann method (LBM), and the cell dynamics was governed by the Newton’s law of translation and rotation. The adhesive dynamics model involved the effect of receptor-ligand bonds between circulating cells and endothelial cells (ECs). It is found that the curved vessel would increase the simultaneous bond number, and the probability of cell adhesion is increased consequently. The interaction between traveling cells would also affect the cell adhesion significantly. For two-cell case, the simultaneous bond number of the rear cell is increased significantly, and the curvature of microvessel further enhances the probability of cell adhesion.     

Effects of wall shear stress and its gradient on tumor cell adhesion in curved microvessels

Tumor cell adhesion to vessel walls in the microcirculation is one critical step in cancer metastasis. In this paper, the hypothesis that tumor cells prefer to adhere at the microvessels with localized shear stresses and their gradients, such as in the curved microvessels, was examined both experimentally and computationally. Our in vivo experiments were performed on the microvessels (post-capillary venules, 30–50 μm diameter) of rat mesentery. A straight or curved microvessel was cannulated and perfused with tumor cells by a glass micropipette at a velocity of ~1mm/s. At less than 10 min after perfusion, there was a significant difference in cell adhesion to the straight and curved vessel walls. In 60 min, the averaged adhesion rate in the curved vessels (n = 14) was ~1.5-fold of that in the straight vessels (n = 19). In 51 curved segments, 45% of cell adhesion was initiated at the inner side, 25% at outer side, and 30% at both sides of the curved vessels. To investigate the mechanical mechanism by which tumor cells prefer adhering at curved sites, we performed a computational study, in which the fluid dynamics was carried out by the lattice Boltzmann method , and the tumor cell dynamics was governed by the Newton’s law of translation and rotation. A modified adhesive dynamics model that included the influence of wall shear stress/gradient on the association/dissociation rates of tumor cell adhesion was proposed, in which the positive wall shear stress/gradient jump would enhance tumor cell adhesion while the negative wall shear stress/gradient jump would weaken tumor cell adhesion. It was found that the wall shear stress/gradient, over a threshold, had significant contribution to tumor cell adhesion by activating or inactivating cell adhesion molecules. Our results elucidated why the tumor cell adhesion prefers to occur at the positive curvature of curved microvessels with very low Reynolds number (in the order of 10⁻²) laminar flow.     

A comparison of primary cultures of rat cerebral microvascular endothelial cells to rat aortic endothelial cells

Summary A method to culture rat cerebral microvascular endothelial cells (RCMECs) was developed and adapted to concurrently obtain cultures of rat aortic endothelial cells (RAECs) without subculturing, cloning, or “weeding.” The attachment and growth requirements of endothelial cell clusters from isolated brain microvessels were first evaluated. RCMECs required fetal bovine serum to attach efficiently. Attachment and growth also depended on the matrix provided (fibronectin≈laminin>gelatin>poly-d-lysine≈Matrigel>hyaluronic acid≈plastic) and the presence of endothelial cell growth supplement and heparin in the growth medium. Non-endothelial cells are removed by allowing these cells to attach to a matrix that RCMECs attach to poorly (e.g., poly-d-lysine) and then transferring isolated endothelial cell clusters to fibronectin-coated dishes. These cell cultures, labeled with 1,1′-dioctadecyl-3,3,3′,3′-tetramethyl-indocarboxyamine perchlorate (DiI-Ac-LDL) and analyzed using flow cytometry, were 97.7±2.6% (n=6) pure. By excluding those portions designed to isolate brain microvessels, the method was adapted to obtain RAEC cultures. RAECs do not isolate as clusters and have different morphology in culture, but respond similarly to matrices and growth medium supplements. RCMECs and RAECs have Factor VIII antigen, accumulate DiI-Ac-LDL, contain Weibel-Palade bodies, and have complex junctional structures. The activities of γ-glutamyl transferase and alkaline phosphatase were measured as a function of time in culture. RCMECs had higher enzymatic activity than RAECs. In both RCMECs and RAECs enzyme activity decreased with time in culture. The function of endothelial cells is specialized depending on its location. This culture method allows comparison of two endothelial cell cultures obtained using very similar culture conditions, and describes their initial characterization. These cultures may provide a model system to study specialized endothelial cell functions and endothelial cell differentiation. This work was funded by the National Institutes of Health grant RO1-NS-21076, and AHA-GIA 881134. Support for Ellen Gordon provided by the National Institutes of Health, NSO7144 and the Seattle Affiliate of the AHA (88-WA-111, 89-WA-112).     

Competition and natural selection in a mathematical model of cancer

A malignant tumor is a dynamic amalgamation of various cell phenotypes, both cancerous (parenchyma) and healthy (stroma). These diverse cells compete over resources as well as cooperate to maintain tumor viability. Therefore, tumors are both an ecological community and an integrated tissue. An understanding of how natural selection operates in this unique ecological context should expose unappreciated vulnerabilities shared by all cancers. In this study I address natural selection’s role in tumor evolution by developing and exploring a mathematical model of a heterogenous primary neoplasm. The model is a system of nonlinear ordinary differential equations tracking the mass of up to two different parenchyma cell types, the mass of vascular endothelial cells from which new tumor blood vessels are built and the total length of tumor microvessels. Results predict the possibility of a hypertumor—a focus of aggressively reproducing parenchyma cells that invade and destroy part or all of the tumor, perhaps before it becomes a clinical entity. If this phenomenon occurs, then we should see examples of tumors that develop an aggressive histology but are paradoxically prone to extinction. Neuroblastoma, a common childhood cancer, may sometimes fit this pattern. In addition, this model suggests that parenchyma cell diversity can be maintained by a tissue-like integration of cells specialized to provide different services.     

Growth of myoblasts in lipoprotein-supplemented,serum-free medium: Regulation of proliferation by acidic and basic fibroblast growth factor

Summary BC₃H1 myoblast cells seeded at low density on gelatin-coated dishes and exposed to a 1∶1 (vol/vol) mixture of Dulbecco’s modified Eagle’s medium and Ham’s F12 medium, proliferate actively when exposed to high density lipoproteins (HDL), transferrin, insulin, and basic or acidic fibroblast growth factor (FGF). This serum-free medium combination supported cell multiplication at a rate equal to that of serum-supplemented medium, and at low cell input (10³ cells/35-mm dish). It also allowed serial transfer of the cultures under serum-free conditions. HDL seems to promote cell survival and to act as progression factor allowing cells to divide when exposed to either basic or acidic FGF. When the potency of basic and acidic FGF were compared, acidic FGF was 20-fold less potent than basic FGF.     

Cerebral microvessels and derived cells in tissue culture: isolation and preliminary characterization.

Microvessels isolated from mouse forebrain were used as the source material for the derivation of cerebral vascular endothelium and smooth-muscle cells in culture. The microvessels were isolated by a mechanical dispersion and filtration technique, and were maintained in vitro as organoid cultures. A microvessel classification system was developed and proved to be useful as a tool in monitoring culture progress and in predicting the type(s) of microvessel(s) that would give rise to migrating and/or proliferating cells. The isolated cerebral microvessels were heterogeneous in diameter, size of individual vascular isolate, and proliferative potential. The isolated microvessels ranged in diameter from 4 micron to 25 micron and in size from a single microvascular segment to a large multibranched plexus with mural cells. The initial viability, determined by erythrosin B exclusion, was approximately 50% on a per cell basis. All microvessel classes had proliferative potential although the rate and extent of proliferation were both microvessel class- and density-dependent. The smaller microvessels gave rise to endothelial cells, whereas the large microvessels gave rise to endothelial and smooth-muscle cells. The viability and progress of a microvessel toward derived cell proliferation seemed to be directly proportional to the number of mural cells present.     

Activation of Rac-1 and Cdc42 stabilizes the microvascular endothelial barrier

We have demonstrated previously that the Rho family GTPase Rac-1 is required for maintenance of endothelial barrier functions in mouse microvascular myocardial endothelial (MyEnd) cells in vitro as well as in rat mesenteric microvessels in vivo. In this study, we tested the hypothesis that specific activation of Rac-1 would stabilize microvascular endothelial barrier functions. For this purpose we used Escherichia coli Cytotoxic necrotizing factor (CNF-1) under conditions (300 ng/ml, 120 min) where it strongly activated Rac-1 and Cdc42 but not Rho A in MyEnd cells. Under these conditions, CNF-1 induced translocation of the actin-binding proteins cortactin and vasodilator-stimulated phosphoprotein (VASP) to cell junctions, increased the junction-associated actin filament belt, and reduced monolayer permeability. We also tested the effect of CNF-1 on endothelial barrier properties in vivo using single-perfused mesenteric microvessels. In contrast to cultured microvascular monolayers, CNF-1 did not reduce baseline barrier functions assayed as hydraulic conductivity (Lp). However, following 120 min pretreatment, CNF-1 significantly attenuated the peak Lp increase in response to platelet-activating factor (PAF, 10 nM) to 12.6±4×10⁻⁷ cm/(s cmH₂O) compared to 46.2±10×10⁻⁷ cm/(s cmH₂O) in experiments using PAF alone. These experiments indicate that activation of Rac-1 and Cdc42 stabilizes microvascular endothelial barrier functions in vitro and in vivo, likely by increasing the junction-associated actin cytoskeleton.     

缺血再灌后血管内皮空泡的形成与内皮的损伤

目的：在活体上探讨缺血再灌后血灌内上细胞损伤及白细胞、血小板与内皮之间粘附的变化。方法：用失血及与再回输血液造成缺血再灌流模型,在高倍显微镜下观察肠系膜微血管内皮损伤及血细胞粘附的变化。结果：缺血再灌后1－3h细静脉、集合毛细血管内出现白细胞、血小板的粘附,血管内皮水肿、管壁增厚,有的血管内皮细胞的胞浆形成圆丘形的空泡,空泡从血管内皮突入管胺、空泡直径10－30μm多出现的细动脉内,在同一根血管内可同时出现几个空泡,大的空泡几科占据血管腔的2／3。结论：缺血再灌后血管内皮水肿及空泡形成,显示内皮细胞的严重损伤。     

In Vitro Recapitulation of Functional Microvessels for the Study of Endothelial Shear Response,Nitric Oxide and [Ca2+]i

Microfluidic technologies enable in vitro studies to closely simulate in vivo microvessel environment with complexity. Such method overcomes certain constrains of the statically cultured endothelial monolayers and enables the cells grow under physiological range of shear flow with geometry similar to microvessels in vivo. However, there are still existing knowledge gaps and lack of convincing evidence to demonstrate and quantify key biological features of the microfluidic microvessels. In this paper, using advanced micromanufacturing and microfluidic technologies, we presented an engineered microvessel model that mimicked the dimensions and network structures of in vivo microvessels with a long-term and continuous perfusion capability, as well as high-resolution and real-time imaging capability. Through direct comparisons with studies conducted in intact microvessels, our results demonstrated that the cultured microvessels formed under perfused conditions recapitulated certain key features of the microvessels in vivo. In particular, primary human umbilical vein endothelial cells were successfully cultured the entire inner surfaces of the microchannel network with well-developed junctions indicated by VE-cadherin staining. The morphological and proliferative responses of endothelial cells to shear stresses were quantified under different flow conditions which was simulated with three-dimensional shear dependent numerical flow model. Furthermore, we successfully measured agonist-induced changes in intracellular Ca²⁺ concentration and nitric oxide production at individual endothelial cell levels using fluorescence imaging. The results were comparable to those derived from individually perfused intact venules. With in vivo validation of its functionalities, our microfluidic model demonstrates a great potential for biological applications and bridges the gaps between in vitro and in vivo microvascular research.     

Young’s modulus of elasticity of Schlemm’s canal endothelial cells

Schlemm’s canal (SC) endothelial cells are likely important in the physiology and pathophysiology of the aqueous drainage system of the eye, particularly in glaucoma. The mechanical stiffness of these cells determines, in part, the extent to which they can support a pressure gradient and thus can be used to place limits on the flow resistance that this layer can generate in the eye. However, little is known about the biomechanical properties of SC endothelial cells. Our goal in this study was to estimate the effective Young’s modulus of elasticity of normal SC cells. To do so, we combined magnetic pulling cytometry of isolated cultured human SC cells with finite element modeling of the mechanical response of the cell to traction forces applied by adherent beads. Preliminary work showed that the immersion angles of beads attached to the SC cells had a major influence on bead response; therefore, we also measured bead immersion angle by confocal microscopy, using an empirical technique to correct for axial distortion of the confocal images. Our results showed that the upper bound for the effective Young’s modulus of elasticity of the cultured SC cells examined in this study, in central, non-nuclear regions, ranged between 1,007 and 3,053 Pa, which is similar to, although somewhat larger than values that have been measured for other endothelial cell types. We compared these values to estimates of the modulus of primate SC cells in vivo, based on images of these cells under pressure loading, and found good agreement at low intraocular pressure (8–15 mm Hg). However, increasing intraocular pressure (22–30 mm Hg) appeared to cause a significant increase in the modulus of these cells. These moduli can be used to estimate the extent to which SC cells deform in response to the pressure drop across the inner wall endothelium and thereby estimate the extent to which they can generate outflow resistance.     

Cerebral microvessels and derived cells in tissue culture

Summary An endothelial cell line has been established from a primary culture of cerebral microvessels isolated from Swiss-Webster mice. The microvessels were isolated by a mechanical dispersion and filtration technique. The cells that emerged from these microvessels, maintained in organoid cultures, proliferated and formed plaques of a single or mixed cell type. The endothelial cell line, designated ME-2, was isolated from one such morphologically homogeneous cell plaque, using both cloning ring techniques and C₆ glioma-conditioned medium. An endothelial specific antiserum was made in rabbits and was used immunocytochemically to confirm the cell type of origin of the ME-2 cell line. Not only did the cell type specific antiserum react exclusively with endothelial cells in vivo, but in the brain the antiserum localized preferentially to the luminal membrane of the endothelium. The ME-2 endothelial cells have retained several of their unique properties such as cytomorphology, growth characteristics, and cell type specific surface antigens throughout the life of the line (in one case 40 passages before senescence). This work was supported in part by an Arteriosclerosis Specialized Center of Research grant from the National Heart, Lung and Blood Institute, National Institutes of Health, Grant HL-14230, and Grant 584-127703 from the Veterans Adminsitration. This paper is dedicated to the memory of Steve Frommes, Electron Microscopist and Photographer.     

Modulation of venular microvessel permeability by calcium influx into endothelial cells.

It has been proposed that calcium ion influx into endothelial cells modulates the permeability of venular microvessels via a calcium-dependent contractile process. The results of recent investigations using permeabilized endothelial cell monolayers conform to this hypothesis by demonstrating a calcium-dependent interaction of endothelial actin and myosin during the retraction of adjacent endothelial cells exposed to inflammatory agents. Little is known about the pathway for calcium influx into endothelial cells after exposure to mediators of inflammation, but evidence suggests that the properties of the calcium entry pathways are similar to the calcium entry pathways that regulate the release of endothelium-derived relaxing factor (EDRF). Substances that stimulate EDRF release from arterial endothelium also increase venular microvessel permeability. Recently developed methods to measure cytoplasmic calcium concentration in the endothelial cells forming the walls of individually perfused microvessels enable a direct investigation of the modulation of the permeability of venular microvessels by calcium influx. These experiments demonstrate that the magnitude of the initial increase in the permeability of microvessels after exposure to an agent that increases permeability, such as a calcium ionophore, is determined by the magnitude of calcium ion influx into the endothelial cells. Furthermore, the magnitude of the calcium influx into endothelial cells is modulated by the membrane potential of the endothelial cells. Depolarization of the endothelial cell membrane reduces calcium influx and attenuates increases in permeability whereas hyperpolarization of the endothelial membrane increases calcium influx and potentiates increases in permeability. These data conform to the hypothesis that a passive conductance channel for calcium is a major pathway for calcium ion flux responsible to eliciting an increase in the permeability of the endothelial barrier in microvessels.     

RGD-avidin–biotin pretargeting to α<Subscript>v</Subscript>β<Subscript>3</Subscript> integrin enhances the proapoptotic activity of TNFα related apoptosis inducing ligand (TRAIL)

Recombinant TNF-related apoptosis-inducing ligand (TRAIL) is considered a powerful and selective inducer of tumor cell death. We hypothesize that TRAIL’s potential as anticancer agent can be enhanced further by promoting its accumulation in tumor tissue. For this purpose, we developed TRAIL complexes that bind to angiogenic endothelial cells. We employed an avidin–biotin pretargeting approach, in which biotinylated TRAIL interacted with RGD-equipped avidin. The assembled complexes killed tumor cells (Jurkat T cells) via apoptosis induction. Furthermore, we demonstrated that the association of the RGD-avidin-TRAIL complex onto endothelial cells enhanced the tumor cell killing activity. Endothelial cells were not killed by TRAIL nor its derived complexes. Our approach can facilitate the enrichment of TRAIL onto angiogenic blood vessels, which may enhance intratumoral accumulation. Furthermore, it offers a versatile technology for the complexation of targeting ligands with therapeutic recombinant proteins and by this a novel way to enhance their specificity and activity.     

Optimization of culture conditions for human corneal endothelial cells

Summary Long-term cultivation of human corneal endothelial cells (HCEC) was optimized with respect to different components of the culture system: 25 different nutrient media, different sera, 6 mitogens and various substrates were tested in their ability to influence clonal growth and morphology of HCEC. F99, a 1∶1 mixture of the two media M199 and Ham’s F12, was the most effective basal medium in promoting clonal growth of HCEC. Among various sera, human serum and fetal bovine serum showed optimal growth promoting activities in combination with F99, whereas newborn bovine serum (NBS) was by far superior for the development of a typically corneal endothelial morphology. Crude fibroblast growth factor (FGF), or alternatively endothelial cell growth supplement, was absolutely essential for clonal growth of HCEC at low serum concentrations, for example 5% NBS. Formation of a monolayer with a morphology similar to corneal endothelium in vivo was observed only on culture dishes coated with basal membrane components such as collagen type IV, laminin, or fibronectin. The most pronounced effect on morphologic appearance was obtained by culturing the cells on the extracellular matrix (ECM) produced by bovine corneal endothelial cells. Moreover, ECM could substitute for crude FGF in clonal growth assays.     

Cellular responses to etoposide: cell death despite cell cycle arrest and repair of DNA damage

The topoisomerase IIα inhibitor etoposide is a ‘broad spectrum’ anticancer agent and a potent inducer of DNA double strand breaks. DNA damage response of mammalian cells usually involves cell cycle arrest and DNA repair or, if unsuccessful, cell death. We investigated these processes in the human colon cancer cell line HT-29 treated with three different etoposide regimens mimicking clinically relevant plasma concentrations of cancer patients. Each involved a period of drug-free incubation following etoposide exposure to imitate the decline of plasma levels between the cycles of chemotherapy. We found a massive induction of double strand breaks that were rapidly and nearly completely fixed long before the majority of cells underwent apoptosis or necrosis. An even greater percentage of cells lost clonogenicity. The occurrence of double strand breaks was accompanied by a decrease in the levels of Ku70, Ku86 and DNA-PK_cs as well as an increase in the level of Rad51 protein. Twenty-four hours after the first contact with etoposide we found a pronounced G₂/M arrest, regardless of the duration of drug exposure, the level of double strand breaks and the extent of their repair. During the subsequent drug-free incubation period, the loss of clonogenicity correlated well with the preceding G₂/M arrest as well as with the amount of cell death found several days after exposure. However, it correlated neither with early apoptosis or necrosis nor with any of the other investigated parameters. These results suggest that the G₂/M arrest is an important determinant in the cytostatic action of etoposide and that the removal of DNA double strand breaks is not sufficient to ensure cell survival.