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Biomedical subjects

U S Ryan

Publications and source records attributed to U S Ryan.

At least 109 records · Page 6Linked to original sources
PubMed1986

Pulmonary endothelium: a dynamic interface.

Appreciation of the cell biology of endothelial cells has contributed greatly to understanding of endothelium as a tissue. Endothelium can no longer be considered simply as an inert barrier with fixed permeabilities, nor as an unreactive expanse of non-thrombogenic surface. Pulmonary endothelium is now recognized to be a tissue composed of metabolically active, functionally responsive cells, that interact with circulating substrates and formed elements in ways that regulate the composition of systemic arterial blood, affect target organ functions, and contribute to thrombosis, hemostasis and immune reactions. Much impetus for the new appreciation of endothelial cell structures and functions has been derived from the ability to isolate and culture pulmonary endothelial cells. Improvements in culture conditions and the growing awareness that biochemical, ultrastructural, as well as physical (fluid mechanical), considerations must be taken into account should pave the way for greater understanding of the role of the endothelium in health and disease.

5'-Nucleotidase↗
PubMed1986

Direct effects of E coli endotoxin on structure and permeability of pulmonary endothelial monolayers and the endothelial layer of intimal explants.

The direct structural, metabolic, and physiologic effects of Escherichia coli endotoxin on bovine pulmonary endothelial monolayers and on the intact endothelial layer of bovine pulmonary artery intimal explants were examined. Endothelial monolayers exposed to E coli endotoxin (0.001, 0.01, 0.1, 1.0, and 10 micrograms/ml) for 24 hours in the absence of bovine fetal calf serum (FCS) showed a dose-dependent response, as demonstrated by number of pyknotic cells and lactate dehydrogenase release that was enhanced by addition of FCS. Prostacyclin production was increased only in the presence of FCS. Endotoxin also caused an increase in permeability. Endothelial cells on nitrocellulose filters placed in chemotaxis chambers with radioactive tracers in the upper well showed a significant 25% increase in rate of equilibration (counts in lower well/counts in upper well) of 3H-water after 2 and 3 hours' incubation with endotoxin (3 hours' endotoxin = 0.89 +/- 0.03 m +/- SE; no endotoxin = 0.69 +/- 0.05) and a 40% increase in equilibration of 125I-albumin at three hours (3 hours' endotoxin = 0.40 +/- 0.03; no endotoxin = 0.27 +/- 0.02). An increase in hydraulic conductance was also seen at 1 hour. Electron microscopy of the endothelial layer of intimal explants showed dilatations in the intercellular junctions and cellular changes representing contraction--increased prominence of cytoplasmic filaments, nuclear crenation, and cytoplasmic protrusions--at 30 and 60 minutes. From 2 hours evidence of cell death was found. Thus, endotoxin causes structural and metabolic changes in pulmonary endothelial cells and an increase in permeability of the endothelial layer. The injury occurs in the absence of FCS but is enhanced by its addition.

Animals↗
PubMed1985

Varicose veins as a source of adult human endothelial cells.

Endothelial cells can be harvested from segments of adult human saphenous vein in a varicose condition removed from patients having single or bilateral vein ligation and stripping. The cells are harvested by scraping with a scalpel, seeded on to gelatin coated or Primaria flasks and are passaged by removal with a rubber policeman. The cells cultured in this manner are maintained in a growth medium that is not supplemented with growth factors. The cells grow with a cobblestone monolayer morphology, possess angiotensin converting enzyme activity and react with antibodies to Factor VIII antigen. The cells fluoresce brightly after reaction with monoclonal antibodies specific for human endothelial cells. Thus, stripped varicose vein segments provide a readily available source of endothelial cells.

Adult↗
PubMed1985

Pulmonary endothelial cell killing by human neutrophils. Possible involvement of hydroxyl radical.

Human blood neutrophils stimulated by a variety of agents were shown to have cytotoxic effects on bovine pulmonary artery endothelial cells. Effective agonists included immune complexes, opsonized zymosan and 12-O-tetradecanoyl phorbol acetate. Unstimulated human neutrophils and neutrophils stimulated with N-formyl-methionyl-leucyl-phenylalanine or with platelet-activating factor failed to induce significant killing even though secretory release of lysosomal enzymes occurred. In comparing the effects of the different agonists, endothelial cell killing showed a better correlation with the production of H2O2 than with the generation of O2-. Endothelial cell killing by stimulated human neutrophils was inhibited by catalase but not by soybean trypsin inhibitor or superoxide dismutase. Killing was also inhibited by two scavengers (N, N-dimethylthiourea and D-mannitol) of hydroxyl radical and by deferoxamine mesylate, an iron-chelator. Iron-saturated deferoxamine mesylate was significantly less effective in protecting the endothelial cells against killing. Agents that were protective against endothelial cell killing did not interfere with the generation of O2- in stimulated neutrophils. These results suggest that leukocyte-induced endothelial cell killing in vitro can be induced by some but not all agonists for neutrophils and that the killing is oxygen-dependent and may be directly due to hydroxyl radical production.

Animals↗
PubMed1985

The pulmonary endothelial surface.

The understanding of endothelial metabolic properties has increased dramatically in recent years. Endothelial cells (ECs) process hormones, drugs, and many blood-borne substances by means of enzymes and transport processes. In turn, some hormones, blood cells, and cellular products interact with ECs via specific receptors on the luminal surface. Functional complexity is exemplified by the metabolism of the adenine nucleotides. ATP, ADP, and AMP are metabolized by enzymes of the endothelial surface to release adenosine, which may be immediately taken up into endothelium and reincorporated intracellularly into nucleotides. Equally complex is the metabolism of the kinins and angiotensins by ECs. Bradykinin is inactivated whereas angiotensin I is converted to angiotensin II. Bradykinin not thus degraded can act on endothelial receptors and stimulate the release of prostacyclin (PGI2). Thus bradykinin can amplify the release of another vasodilator, PGI2, and can stimulate the release of a powerful antiaggregatory agent (PGI2). Many of these complex metabolic reactions occur at the endothelial surface, a structure that is itself complex. ECs possess endothelial projections and caveolae as well as a fuzzy coat, or glycocalyx. Functions of the endothelial glycocalyx are not well understood, but the glycocalyx can now be visualized: it may act as a molecular sieve and provide a substratum for the initiation and progression of immunologic reactions.

Adenosine Diphosphate↗
PubMed1985

Glucocorticoid modulation of prostacyclin production in cultured bovine pulmonary endothelial cells.

The present study has examined the ability of glucocorticoids to modify the production of prostacyclin by endothelial cells derived from bovine pulmonary arteries. Binding studies with [3H]dexamethasone indicated that these cells possessed high-affinity binding sites for glucocorticoids (Kd approximately 4 nM). The cells released prostacyclin, measured serologically as its hydrolysis product 6-keto-prostaglandin F1 alpha. The release was strongly stimulated by a 5-min incubation with 50 nM bradykinin, 2 microM calcium ionophore A23187 or 1 to 5 microM arachidonic acid. Dexamethasone, 1 to 20 nM, suppressed prostacyclin release in response to bradykinin and ionophore in a dose-dependent manner. The suppressive effects required 24 hr for full expression. Maximal inhibition of bradykinin-induced prostacyclin release was approximately 65% and that of ionophore-induced release was approximately 35%. Total inhibition was not observed. Hydrocortisone at 20 nM inhibited bradykinin-induced release of prostacyclin by approximately 45% but had no effect on ionophore-induced release. Neither glucocorticoid inhibited prostacyclin release in response to arachidonic acid. Triton X-100 extracts and conditioned media from cells treated with 20 nM dexamethasone failed to modify prostacyclin release when added to fresh endothelial cells. We conclude that dexamethasone, in concentrations likely to produce virtually complete binding site occupancy, inhibits agonist-induced release of prostacyclin from bovine pulmonary endothelial cells. Inhibition appears to occur at the level of intracellular arachidonic acid release. However, we have been unable to obtain definitive evidence for the production of a macromolecular phospholipase inhibitor by endothelial cells in response to these low concentrations of steroid.

Animals↗
PubMed1984

Adult human saphenous vein endothelial cells: assessment of their reproductive capacity for use in endothelial seeding of vascular prostheses.

Autogenous endothelial seeding (AES) of vascular prostheses (VP) using venous endothelial cells (EC) reduces platelet-VP interactions and improves patency rates in small caliber VP in dogs. To conserve patients' veins for use in coronary or limb bypass surgery, human trials of AES should require proof that adequate numbers of EC with the growth capacity to cover VP can be harvested from acceptably small pieces of peripheral vein. EC were isolated from excess saphenous vein segments remaining after coronary bypass surgery by filling veins with 0.1% CLS II collagenase at 37 degrees C for 15 min and removing EC by flushing the veins with culture medium. EC were cultured on fibronectin-coated dishes in medium 199 with 30% human serum and 300 micrograms/ml of endothelial cell growth factor. These cells grew to form confluent monolayers, and were identified as EC by tests for factor VIII antigen. Veins from 53 patients with a mean age of 55.8 +/- 9.8 (SD) years yielded vein segments with an average area of 1.9 +/- 0.6 cm2, from which an average of 5.3 +/- 2.8 X 10(4) cells were removed per cm2 of vein area. EC in culture underwent 14.3 +/- 1.4 population doublings with an average population doubling time of 1.8 +/- 0.3 days (N = 14 cultures), which allowed an 100-fold increase in cell number to occur in 11 to 12 days. These data suggest that the EC available from small vein segments in adult humans have the growth capacity to cover areas comparable in size to the luminal areas of VP commonly used in arterial surgery.

Adult↗
PubMed1984

Trypsin-induced aggregation of bovine pulmonary artery endothelial cells cultured on microcarriers.

We studied adherence between 'luminal' surfaces of pulmonary artery endothelial cells by standard aggregometry techniques, widely used for measuring aggregation of platelets and granulocytes. Using suspensions of bovine pulmonary artery endothelial cells cultured on microcarrier beads, in an aggregometer, we found that trypsin caused endothelial aggregation. The aggregation response occurred at trypsin concentrations as low as 0.001%. The degree of trypsin-induced aggregation indicated by the magnitude of the change in light transmission through the endothelial suspensions was related to the trypsin concentration, reaching a maximum level at trypsin concentrations of 0.01%. We conclude that trypsin, even in very low concentrations, causes adherence between 'luminal' surfaces of pulmonary endothelial cells probably because the enzyme destroys cell surface proteins which are necessary to prevent intercellular adherence. The method we describe may be useful for studying cell-cell interactions of endothelium.

Animals↗
PubMed1984

Isolation and culture of pulmonary endothelial cells.

Methods for isolation, identification and culture of pulmonary endothelial cells are now routine. In the past, methods of isolation have used proteolytic enzymes to detach cells; thereafter, traditional methods for cell passaging have used trypsin/EDTA mixtures. Cells isolated and passaged using proteolytic enzymes have been useful in establishing the field and in verifying certain endothelial properties. However, there is a growing awareness of the role of endothelial cells in processing vasoactive substances, in responding to hormones and other agonists and in cell-cell interactions with other cell types of the vascular wall, with blood cells and with cellular products. Consequently, a new requirement has arisen for cells in vitro that maintain the differentiated properties of their counterparts in vivo. The deleterious effects of trypsin and other proteolytic enzymes commonly used in cell culture on surface structures of endothelial cells such as enzymes, receptors and junctional proteins, as well as on extracellular layers such as the glycocalyx or "endothelial fuzz," have led to the development of methods that avoid use of proteolytic enzymes at both the isolation step and during subsequent subculture. This chapter describes traditional methods for isolating pulmonary endothelial cells but emphasizes newer approaches using mechanical harvest and scale-up using microcarriers. The new methods allow maintenance of long-term, large-scale cultures of cells that retain the full complement of surface properties and that maintain the cobblestone monolayer morphology and differentiated functional properties. Methods for identification of isolated cells are therefore also considered as methods for validation of cultures during their in vitro lifespan.

Animals↗
PubMed1984

The ultrastructural basis of endothelial cell surface functions.

Several enzymes, enzyme inhibitors, receptors and transport structures are situated on the luminal surface of endothelium. Some enzymes and transport systems are continuously active and, in effect, regulate the composition of blood moving downstream. Other components are latent. Their activities are not expressed in the absence of stimulus. Thus, endothelial cells injured by granulocytes or viral infection possess receptors for the Fc segment of IgG and for C3b, whereas normal endothelial cells do not. Both normal and injured endothelial cells express receptors for Clq. To visualize surface enzymes, inhibitors, receptors and transport structures, we have prepared surface replicas suitable for high resolution EM. The surface replication technique, coupled with immunocytochemical procedures, facilitates studies of the topographies of surface enzymes such as angiotensin converting enzyme (ACE) and carboxypeptidase N (CPN). In addition, the replicas provide unique views of the glycocalyx, a cell coating previously believed to be amorphous but now seen to be a highly organized carpet-work under which surface enzymes, receptors, etc are embedded. Given its content of fibronectin, the glycocalyx may be the Clq receptor. Cells treated with antibodies to ACE or CPN show disarrayed glycocalyces and bind Fc and C3b. Latency of the Fc and C3b receptors may be owing to the physical barrier provided by the glycocalyx. Apparently, damage to the glycocalyx creates conditions favoring binding of immune complexes, complement activation and intravascular coagulation with loss of gradients between blood and parenchyma. Whether some non-thrombogenic properties of endothelium require an intact glycocalyx deserves consideration as does the role of the glycocalyx in regulating microvascular permeability.

Animals↗
PubMed1984

Cell biology of pulmonary endothelium.

Since the late 1960s, understanding of the cell biology of endothelium has been transformed. Endothelium is not merely a metabolically inert, semipermeable barrier separating blood from parenchyma; rather, it is a layer of metabolically active cells. In lungs, even gas exchange may be assisted by reactions occurring on the endothelial surface. Endothelial cells synthesize specific proteins (some for export); these cells have receptors and enzymes capable of reacting with certain hormones and other excitatory substances as they pass in circulating blood. Endothelium is antithrombogenic unless injured; when injured, endothelium becomes thrombogenic and then thrombolytic. Endothelium may sometimes retard the development of inflammation and at other times may facilitate it. In addition to providing sites for exchange of nutrients and metabolites, endothelium interacts with prohormones and hormones to determine the composition of blood moving downstream. The latter is a key function of pulmonary endothelium: its venous effluent is systemic arterial blood. Efforts to understand how endothelium accomplishes its wide range of metabolic activities have motivated parallel efforts to define the fine structure of the endothelial cell. Thus it has become feasible to visualize habitats of two surface enzymes, angiotensin-converting enzyme and carboxypeptidase N. Efforts to visualize surface enzymes required development of means of replicating cell surfaces, a methodology that in turn provided the first en face view of the glycocalyx. Given the ubiquity of vascular endothelium and its activities, it is difficult to imagine an area of medical practice that can safely ignore requirements for appropriately functioning endothelium.

Cardiology↗
PubMed1983

Bradykinin-induced release of prostacyclin and thromboxanes from bovine pulmonary artery endothelial cells. Studies with lower homologs and calcium antagonists.

Bovine pulmonary artery endothelial cells, in serum-free culture medium, release small quantities of prostacyclin and thromboxane A2 (3-10 and 0.1-0.3 ng/ml; measured as immunoreactive 6-ketoprostaglandin F1 alpha and thromboxane B2, respectively). The release of these substances is stimulated by up to 20-fold during a 3 min incubation with the vasodilator, bradykinin (Arg1-Pro2-Pro3-Gly4-Phe5-Ser6-Pro7-Phe8-Arg9). Endothelial cells incubated with [3H]arachidonic acid for 24 h and then exposed to bradykinin for 3 min release 3H into the medium, approximately 65% of which co-chromatographs with 6-ketoprostaglandin F1 alpha and 3% with thromboxane B2. The effects of bradykinin are dose-related and are often discernible when the hormone is used at concentrations believed to occur physiologically (10 pg/ml; approximately 10 pM). Furthermore, the bradykinin molecule must be intact: none of its lower homologs affects the release of prostacyclin, thromboxane A2, or 3H unless used at concentrations (1 microM or higher) unlikely to be achieved in vivo. The release appears to involve calcium uptake and calmodulin: it is abolished by EGTA (5 mM) and inhibited by the 'slow channel' calcium antagonists, verapamil and nifedipine (10-100 microM), and by the calmodulin inhibitor, trifluoperazine (3-30 microM). Our findings suggest that bradykinin exerts some of its hormonal effects by acting on specific receptors possessed by vascular endothelial cells; receptor activation is associated with calcium transport, arachidonate mobilization, and a selective synthesis of prostacyclin, a vasodilator in its own right.

Animals↗