Beta-adrenergic receptor gene polymorphisms and hemodynamic response to dobutamine during dobutamine stress echocardiography.

Our objective was to determine if beta(1)-adrenergic receptor (beta(1)-AR) and beta(2)-AR gene polymorphisms influence heart rate (HR), systolic blood pressure (SBP) and diastolic blood pressure (DBP) response to dobutamine during dobutamine stress echocardiography (DSE). Patients (n=163) undergoing clinically indicated DSE were enrolled. Dobutamine doses were titrated from 5 to 40 microg kg(-1) min(-1) at 3 min intervals and HR, SBP and DBP were measured. Genotypes were determined for beta(1)-AR Ser49Gly, beta(1)-AR Arg389Gly, beta(2)-AR Arg16Gly and beta(2)-AR Gln27Glu polymorphisms by polymerase chain reaction-restriction fragment length polymorphism analysis, pyrosequencing and single primer extension methods. beta(2)-AR Glu27 homozygotes had a greater HR response at the highest dobutamine dose than Gln27 carriers (P=0.002). Beta(2)-AR Gly16 homozygotes had a lower HR response during 5-30 microg kg(-1) min(-1) of the dobutamine infusion protocol compared to Arg16 carriers (P=0.03). Differences in SBP by beta(2)-AR codon 16 genotype and DBP by beta(1)-AR codon 389 genotype were found at baseline and were maintained throughout DSE (P=0.06 and 0.02, respectively). However, the magnitude of SBP and DBP response to dobutamine did not differ significantly by beta(2)-AR codon 16 or beta(1)-AR codon 389 genotypes, respectively. These data suggest that the four selected beta(1)- and beta(2)-AR polymorphisms do not substantially influence the magnitude of hemodynamic response to dobutamine during DSE.

The terminal complement proteins C5b-9 augment binding of high density lipoprotein and its apolipoproteins A-I and A-II to human endothelial cells.

Terminal complement protein complexes C5b-9 have been found in human atherosclerotic lesions. Insertion of C5b-9 in the endothelial cell membrane alters permeability, induces membrane vesiculation, and activates secretion. We hypothesized that complement might also alter interactions of the endothelial surface with lipoproteins, particularly high density lipoprotein (HDL), which is reported to inhibit C5b-9-induced hemolysis. We now demonstrate that exposure to C5b-9 increases (by 2- to 50-fold) specific binding of HDL and its apolipoproteins (apo) A-I and A-II to endothelial cells. Binding to cells exposed to antibody, C5b67, and C5b-8 was virtually unchanged. Enhanced binding was also dependent on the number of C5b-9 complexes deposited on the cells. Other agonists that activate endothelial secretion did not augment binding. Calcium was required for full exposure of new binding sites by C5b-9. The C5b-9-induced increase in binding was independent of the increase observed after cholesterol loading. In addition, apo A-I and A-II appear to compete for the same binding sites on untreated and C5b-9-treated cells. In contrast to the data reported for red cells, we were unable to detect significant inhibition of C5b-9-mediated endothelial membrane permeabilization by HDL (up to 1 mg/ml) or by apo A-I (up to 100 micrograms/ml). These data demonstrate that the C5b-9 proteins enhance endothelial binding of HDL and its apoproteins, suggesting that intravascular complement activation may alter cholesterol homeostasis in the vessel wall.

Changes in cytosolic Ca2+ associated with von Willebrand factor release in human endothelial cells exposed to histamine. Study of microcarrier cell monolayers using the fluorescent probe indo-1.

A method for measuring fluorescence in anchored monolayers of human endothelial cells is described and used to demonstrate changes in the cytosolic free-calcium concentration ([Ca2+]c) in these cells exposed to histamine and thrombin; some endothelial responses to both agonists (e.g., mitogenesis) have been suggested to be Ca2+-mediated. Umbilical vein endothelial cells were cultured on microcarriers and loaded with the Ca2+ indicator, indo-1. Enzymatic cell detachment was avoided by monitoring the indo-1 fluorescence ratio (400/480 nm) of a stirred suspension of cell-covered microcarriers. Basal [Ca2+]c was estimated to be 70-80 nM. Thrombin induced a transient dose-dependent increase in [Ca2+]c, which was active-site dependent. Histamine stimulated a dose-dependent increase in [Ca2+]c, which was reversed by removal of histamine and inhibited competitively by the H1-receptor antagonist pyrilamine, but not by the H2-receptor antagonist cimetidine. Furthermore, histamine induced a dose-dependent secretion of von Willebrand factor, which paralleled the rise in [Ca2+]c and was similarly blocked by the H1-receptor antagonist, and which may contribute to platelet deposition at sites of inflammation.

Effects of plasmin on von Willebrand factor multimers. Degradation in vitro and stimulation of release in vivo.

von Willebrand factor (vWF), a multimeric protein that mediates platelet adhesion, circulates in association with the procoagulant Factor VIII (FVIII). In previous reports, plasmin was shown in vitro to inactivate FVIII and cleave the vWF subunit extensively, but to cause only a modest decrease in vWF platelet-agglutinating activity. In the present study, the digestion of vWF multimers by plasmin was analyzed by sodium dodecyl sulfate-agarose gel electrophoresis and radioimmunoblotting. In vitro, plasmin degraded the large vWF multimers to smaller forms that could be distinguished from the small multimers present before digestion only by a slightly increased electrophoretic mobility. These plasmin-cleaved "multimers" were composed of disulfide-linked fragments with no intact vWF subunits. Thus, many plasmin cleavages occur within disulfide loops. The slight increase in mobility of plasmin-digested vWF is in part explained by the early cleavage from the multimers of a 34,000-mol wt peptide, which was purified and partially sequenced. The amino-terminal sequence (33 residues) agrees with the previously reported sequence (15 residues) for the amino terminus of the intact vWF subunit. Analysis of plasmin-digested vWF allowed deduction of a model for the native vWF structure, including the approximate location of the interprotomer disulfide bond(s). To determine whether plasmin would digest vWF in vivo, plasmas from 12 patients and 2 normal volunteers who received intravenous streptokinase (SK) were analyzed. Rather than vWF digestion, a two- to threefold rise in vWF antigen and platelet-agglutinating activity occurred within 2 h after a single SK dose, and the increase was greatest among the largest multimers. In contrast, FVIII clotting activity dropped to 10-20% of pre-SK levels. Thus, although plasmin destroys FVIII, a pharmacologically induced fibrinolytic state is associated with significant release of vWF from endothelial cells, platelets, or some other storage pool.

Substructure of human von Willebrand factor.

Using electron microscopy, we have visualized the substructure of human von Willebrand factor (vWf) purified by two different approaches. vWf multimers, which appear as flexible strands varying in length up to 2 micron, consist of dimeric units (protomers) polymerized linearly in an end-to-end fashion through disulfide bonds. Examination of small multimers (e.g., one-mers, two-mers, and three-mers) suggests that each protomer consists of two large globular end domains (22 X 6.5 nm) connected to a small central node (6.4 X 3.4 nm) by two flexible rod domains each approximately 34 nm long and approximately 2 nm in diameter. The protomer is 120 nm in length when fully extended. These same structural features are seen both in vWf molecules that were rapidly purified from fresh plasma by a new two-step procedure and in those purified from lyophilized intermediate-purity Factor VIII/vWf concentrates. The 240,000-mol wt subunit observed by gel electrophoresis upon complete reduction of vWf apparently contains both a rod domain and a globular domain and corresponds to one half of the protomer. Two subunits are disulfide-linked, probably near their carboxyl termini, to form the protomer; disulfide bonds in the amino-terminal globular ends link promoters to form vWf multimers. The vWf multimer strands have at least two morphologically distinct types of ends, which may result from proteolytic cleavage in the globular domains after formation of large linear polymers. In addition to releasing fragments that were similar in size and shape to the repeating protomeric unit, plasmic degradation of either preparation of vWf reduced the size of multimers, but had no detectable effect on the substructure of internal protomers.

Cyclosporine suppression of endothelial prostacyclin generation. A possible mechanism for nephrotoxicity.

The effects of cyclosporine (CsA) on prostacyclin (PGI2) release by cultured human umbilical vein endothelial cells were investigated. PGI2 production was measured by radioimmunoassay of its stable metabolite 6-Keto-PGF1 alpha. CsA induced a time and concentration-dependent reduction in unstimulated (basal) and Ca++ ionophore (A23187)-stimulated release of PGI2. A 16-hr incubation with CsA reduced A23187 PGI2 release by 64% (P less than 0.05); CsA at concentrations of 1.0, 10.0, and 100.0 micrograms/ml reduced A23187 PGI2 release by 67%, 80%, and 90%, respectively (P less than 0.05). This suppression was reversed within 24 hr after withdrawal of CsA. Arachidonic acid-stimulated PGI2 release was also decreased in CsA-treated cells, indicating an inhibitory effect distal to phospholipase A2. 3H-deoxyglucose release, an indicator of cell injury, was not increased by CsA, thus excluding nonspecific cell damage as a mechanism of the observed suppressive effect. This inhibition of PGI2 release from endothelial cells by CsA may explain the increased renal vascular resistance and renal microvascular thrombosis seen on occasion with CsA administration.

Anatomically flexible, computer-assisted reporting system for coronary angiography.

A computer-assisted method for reporting coronary arteriographic findings has been developed. A videographic display with a touch input system permits finger pointing to designate arterial segments, lesion locations, graft insertion points and collateral vessels. The coronary diagram is flexible to accommodate variations in dominance and in the size and length of the left coronary system branches. Lesion severity, type and length of lesions, distal vessel anatomy, collateral circulation and coronary bypass grafts (including Y or jump grafts) are displayed pictographically. A tabular summary and graphic output provide a completed coronary report eliminating the need for narrative dictation. An edit mode provides capability for teaching and review. The computer-assisted reporting method has the advantage of automatic data encoding in formats suitable for data base storage and subsequent retrieval.

Antioxidant supplements during cancer treatments: where do we stand?

Chemotherapeutic agents operate through a number of different mechanisms; not all of them depend on ROS for their cytotoxic activity. Drugs with pharmacologic action that do not depend on ROS also are available; these are less susceptible to possible interference by antioxidants. In addition, caution should be exercised with investigational drugs whose mechanisms are being explored (Labriola & Livingston, 1999). Regardless, without studies specifically evaluating the impact that antioxidants have on cancer therapies, one still cannot confidently advise patients with cancer to supplement their diet with large doses of antioxidants for potential anticancer and protectant effects. It has been scientifically supported that antioxidants have a beneficial effect on reducing extraneous oxidative damage; so, based on what is known to date, an antioxidant-rich diet seems to be the most prudent advice when confronted with the advisability of supplementation during cancer therapy. In the near future, it is hoped that studies will further define the relationship between the many different cancer diseases, treatments, and supplemental antioxidants.

Interaction between apolipoproteins A-I and A-II and the membrane attack complex of complement. Affinity of the apoproteins for polymeric C9.

We have previously observed enhanced binding of HDL and apolipoproteins A-I and A-II to human endothelial cells exposed to activated complement. Induction of these binding sites required complement activation through C9, suggesting a specific role for the C9 component of the C5b-9 complex. We now report that specific and saturable binding sites for apoA-I and -A-II are expressed by C9 polymers (polyC9), whereas little binding was observed to native monomeric C9. These data suggested an interaction of the apoproteins with a site(s) which is exposed only upon C9 polymerization, and also suggested that binding of the apoproteins to this new site might interfere with assembly of C9 into the polyC9 tubule and insertion into the cell membrane. ApoA-I was found to inhibit zinc-catalyzed polymerization of C9 in a concentration-dependent fashion. Formation of SDS-resistant C9 polymers was completely inhibited at apoA-I or -A-II concentrations > or = 5 microM. ApoA-I also produced a concentration-dependent inhibition of C9 incorporation into C5b-9 complexes on endothelial cells, which was accompanied by a corresponding decrease in SDS-resistant C9 polymers associated with the cell membrane. In summary, the ability of the HDL apoproteins A-I and A-II to interact with an activation-dependent conformer(s) of the C9 component of the C5b-9 complex appears to explain the expression of HDL binding sites on endothelial cells exposed to complement. These apoproteins are also inhibitors of C9 polymerization, which may underlie the protective effect of HDL for blood cells exposed to activated complement.

A eukaryotic DNA glycosylase/lyase recognizing ultraviolet light-induced pyrimidine dimers.

Cyclobutane pyrimidine dimers (CPDs) are the predominant product of photodamage in DNA after exposure of cells to ultraviolet light and are cytotoxic, mutagenic and carcinogenic in a variety of cellular and animal systems. In prokaryotes, enzymes and protein complexes have been characterized that remove or reverse CPDs in DNA. Micrococcus luteus and T4 phage-infected Escherichia coli contain a specific N-glycosylase/apurinic-apyrimidinic lyase that catalyses a two-step DNA incision process at sites of CPDs, thus initiating base excision repair of these lesions. It is well established that CPDs are recognized and removed from eukaryotic DNA by excision repair processes but very little information exists concerning the nature of the proteins involved in CPD recognition and DNA incision events. We report here that an enzyme functionally similar to the prokaryotic N-glycosylase/apurinic-apyrimidinic lyases exists in Saccharomyces cerevisiae. To our knowledge, this is the first time such an activity has been found in a eukaryote and is also the first example of an organism having both direct reversal and base excision repair pathways for the removal of CPDs from DNA.

Complement C5b-9 increases plasminogen binding and activation on human endothelial cells.

Deposition of the terminal complement proteins (C5b-9) on human endothelial cells can result in cell lysis or nonlytic alterations of cell function including procoagulant responses. Because regulation of fibrinolysis is a central endothelial function and because C9 contains a carboxyl-terminal lysine similar to other proteins that bind and facilitate activation of plasminogen (PG), the effects of complement injury on PG binding and activation on these cells were investigated. Activation of complement through deposition of C5b67 complexes on endothelial cells resulted in a small increase (approximately 20%) in PG binding. Incorporation of C8 into C5b-8 resulted in no further increase in binding; however, specific 125I-PG binding was increased by approximately 100% after C5b-9 deposition. Moreover, PG was found to bind specifically to C7 and C9. The PG bound to endothelial cells after C5b-9 deposition was readily activated by tissue-type plasminogen activator (TPA). In a cell-free system, complement C9 and a synthetic peptide composed of the 20 carboxyl-terminal amino acids of C9 enhanced PG activation by TPA. Removal of the carboxyl-terminal lysine of C9 abolished the enhancement of PG activation without diminishing PG binding. We conclude that membrane C9 may comprise a binding site for PG and serve to enhance activation of this zymogen by TPA. These findings suggest that immune injury to the endothelium may enhance both the fibrin-generating and fibrinolytic capacity of the vessel wall.

Regulatory control of the terminal complement proteins at the surface of human endothelial cells: neutralization of a C5b-9 inhibitor by antibody to CD59.

Functionally inhibitory antibody to the plasma membrane complement inhibitor CD59 has been used to investigate control of the terminal complement proteins at the endothelial cell surface. Antibodies against purified human erythrocyte CD59 (polyclonal anti-CD59 and monoclonal antibodies [MoAbs] 1F1 and 1F5) were found to bind specifically to monolayers of cultured human umbilical vein endothelial cells, and by Western blotting to recognize an 18- to 21-Kd endothelial protein. When bound to the endothelial monolayer, anti-CD59 (immunoglobulin G or Fab fragment) potentiated membrane pore formation induced upon C9 binding to C5b-8, and augmented the C5b-9-induced cellular responses, including stimulated secretion of von Willebrand factor and expression of catalytic surface for the prothrombinase enzyme complex. Although potentiating endothelial responses to the terminal complement proteins, anti-CD59 had no effect on the response of these cells to stimulation by histamine. Taken together, these data suggest that human endothelial cells express the CD59 cell surface inhibitor of the terminal complement proteins, which serves to protect these cells from pore-forming and cell-stimulatory effects of the C5b-9 complex. These data also suggest that the inactivation or deletion of this cell surface regulatory molecule would increase the likelihood for procoagulant changes in endothelium exposed to complement activation in plasma.

Complement proteins C5b-9 initiate secretion of platelet storage granules without increased binding of fibrinogen or von Willebrand factor to newly expressed cell surface GPIIb-IIIa.

The functional and conformational activation of cell surface glycoproteins IIb-IIIa (GPIIb-IIIa) was probed in platelets stimulated to secrete by complement proteins C5b-9. Gel-filtered human platelets exposed to the purified human C5b-9 proteins exhibited non-lytic secretory release of both alpha- and dense granule storage pools with only a small increase in total binding of 125I-fibrinogen (less than 3000 molecules/cell) to the cell surface. By contrast to ADP- or thrombin-activated platelets, increased 125I-fibrinogen bound to C5b-9 platelets was not inhibited by Arg-Gly-Asp-containing peptides, suggesting that the high affinity membrane receptor for fibrinogen is not expressed under these conditions. C5b-9-stimulated platelets also failed to bind 125I-von Willebrand factor (less than 1 ng/10(8) platelets), confirming that the adhesive protein receptor function of cell surface GPIIb-IIIa is not expressed in these cells. Although specific binding of 125I-fibrinogen or 125I-von Willebrand factor did not significantly increase after C5b-9 assembly, these proteins elicited de novo expression of the GPIIb-IIIa activation-associated epitope recognized by monoclonal antibody PAC-1, and binding of this antibody to C5b-9 platelets was fully competed by Arg-Gly-Asp-containing peptides. These data suggest that the metabolic events which trigger granule secretion after C5b-9 insertion into the plasma membrane cause cell surface GPIIb-IIIa to be expressed in an activation-associated but functionally incompetent conformation.

Complement proteins C5b-9 induce vesiculation of the endothelial plasma membrane and expose catalytic surface for assembly of the prothrombinase enzyme complex.

Assembly of the terminal complement proteins C5b-9 on human endothelial cells results in increased cytosolic calcium and nonlytic secretion of high molecular weight multimers of von Willebrand factor from intracellular storage granules. We now demonstrate that this C5b-9-induced secretory response is accompanied by vesiculation of membrane particles from the endothelial surface which express binding sites for factor Va and support prothrombinase activity. Exposure of factor Va binding sites after C5b-9 assembly was accompanied by greater than 2-fold increase in prothrombinase activity, which was not observed for cells exposed to C5b-8 (in the absence of C9). By contrast, only a 3-16% increase in prothrombinase activity was observed when these cells were maximally stimulated to secrete by either histamine, thrombin, or the Ca2+ ionophore A23187. Increased prothrombinase activity after C5b-9 was not accompanied by a change in thrombomodulin activity, and was unrelated to cell lysis, the complement-treated cells remaining greater than 99% viable. Endothelial prothrombinase activity was predominately associated with small membrane vesicles (less than 1 microns diameter) released from the cell monolayer. Analysis by fluorescence-gated flow cytometry revealed that these vesicles incorporate the C5b-9 proteins and express binding sites for factor Va. The capacity of the C5b-9 proteins to induce vesiculation of the endothelial plasma membrane and thereby expose catalytic surface for the prothrombinase enzyme complex may contribute to fibrin deposition associated with immune endothelial injury.

Identification of a mammalian nuclear factor and human cDNA-encoded proteins that recognize DNA containing apurinic sites.

Damage to DNA can have lethal or mutagenic consequences for cells unless it is detected and repaired by cellular proteins. Repair depends on the ability of cellular factors to distinguish the damaged sites. Electrophoretic binding assays were used to identify a factor from the nuclei of mammalian cells that bound to DNA containing apurinic sites. A binding assay based on the use of beta-galactosidase fusion proteins was subsequently used to isolate recombinant clones of human cDNAs that encoded apurinic DNA-binding proteins. Two distinct human cDNAs were identified that encoded proteins that bound apurinic DNA preferentially over undamaged, methylated, or UV-irradiated DNA. These approaches may offer a general method for the detection of proteins that recognize various types of DNA damage and for the cloning of genes encoding such proteins.

Stimulated secretion of endothelial von Willebrand factor is accompanied by rapid redistribution to the cell surface of the intracellular granule membrane protein GMP-140.

We have examined the cell activation-dependent redistribution of the intracellular granule membrane protein GMP-140 of human endothelial cells. By dual-label immunofluorescence, the distribution of GMP-140 within cultured human umbilical vein endothelial cells was found to coincide with the distribution of von Willebrand factor (vWF), suggesting that GMP-140 is located in the membranes of vWF-containing storage granules. Stimulation of vWF secretion resulted in an increase in GMP-140 on the cell surface, as detected by increased binding of the monoclonal antibody S12 which recognizes the extracytoplasmic domain of GMP-140. For each agonist tested (histamine, thrombin, phorbol 12-myristate 13-acetate, and the calcium ionophore A23187) a dose-dependent redistribution of GMP-140 to the endothelial surface was observed which closely paralleled the dose-dependent secretion of vWF into the cell supernatant. When cells were maximally stimulated by histamine in the presence of antibody S12, a 4-fold increase in S12 uptake by the cells was observed. This increase occurred rapidly and reached a plateau by 10 min. In contrast, when histamine-stimulated cells were first fixed with paraformaldehyde or chilled to 4 degrees C before addition of antibody S12, only a transient increase in cell surface GMP-140 was detected. Under these conditions of arrested membrane turnover during antibody binding, cell surface GMP-140 was maximal 3 min after histamine stimulation and then declined to control levels by 20 min. These data suggest that stimulated secretion of vWF from endothelial cells entails fusion of vWF-containing storage granules with the plasma membrane. Once inserted into the plasma membrane, GMP-140 is subsequently removed from the endothelial surface, most likely by an endocytic mechanism.