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.

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.

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.

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.

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.

DNA binding properties of YB-1 and dbpA: binding to double-stranded, single-stranded, and abasic site containing DNAs.

A number of eukaryotic DNA binding proteins have been isolated by screening phage expression libraries with DNA probes containing the binding site of the DNA-binding protein. This methodology was employed here to isolate clones of the factor that interacts with the W box element of the human major histocompatibility complex HLA-DQB gene. Surprisingly, several cDNA clones of YB-1, a cDNA clone that was previously isolated with a CCAAT element-containing sequence were found. Independently, the screening of phage expression libraries with depurinated DNA resulted in the isolation of YB-1 and dbpA, a previously isolated cDNA that has homology to YB-1. Additional characterization of YB-1 showed that it bound a wide variety of DNA sequences and suggested that the binding of this protein is promiscuous. Furthermore, we show that both YB-1 and dbpA bind to depurinated DNA better than undamaged DNA and that the extent of specificity of binding is influenced by Mg2+. Due to the lack of sequence specificity and high degree of binding to depurinated DNA, we suggest that these proteins might be involved in chromosome functions such as maintenance of chromatin structure or DNA repair that do not require sequence-specific binding.

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.

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.

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.

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.

Complement proteins C5b-9 induce secretion of high molecular weight multimers of endothelial von Willebrand factor and translocation of granule membrane protein GMP-140 to the cell surface.

The effect of immune activation of the serum complement system on the secretory response of human endothelial cells was examined. Exposure of antibody sensitized cultured umbilical vein endothelial cells to human serum resulted in secretion of very high molecular weight multimers of von Willebrand factor which coincided with new surface expression of the intracellular granule membrane protein GMP-140. This response required complement activation through deposition of C5b-9 and was not observed with cells exposed to antibody plus C8-deficient serum or to membrane C5b-8 (in the absence of C9). This C5b-9-induced secretion was observed with minimal cell lysis, as assessed by the release of lactic dehydrogenase. Delayed addition of C8 and C9 to cells exposed to antibody plus C8-deficient serum revealed a rapid decay of membrane C8 binding sites accompanied by loss of the secretory response, suggesting a process of removal or inactivation of nascent C5b67 complexes deposited on the endothelial surface. Membrane assembly of C5b-9 complexes caused an increase in endothelial cytosolic [Ca2+], due to influx across the plasma membrane. This C5b-9-dependent increase in cytosolic [Ca2+] and concomitant von Willebrand factor secretion were both abolished by removal of external calcium. In addition to being linked to the level of external Ca2+, the C5b-9-induced secretory response was partially inhibited by the protein kinase inhibitor, sphingosine. The capacity of the C5b-9 proteins to stimulate endothelial cells to secrete a platelet adhesive protein provides one mechanism for increased platelet deposition at sites of inflammation, and suggests the potential for other functional changes in endothelium exposed to C5b-9 during intravascular complement activation.

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.

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.

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.