Inhibition of Cap-initiation complexes linked to a novel mechanism of eIF4G depletion in acute myocardial ischemia.

Translational control in the rat heart was characterized during acute myocardial ischemia introduced by left coronary artery ligature. Within 10 min of ischemia, eukaryotic (eIF)4E binds to its negative regulator, eIF4E-binding protein-1 (4E-BP1), but the levels of 4E-BP1 are insufficient to disrupt cap-dependent mRNA initiation complexes. However, by 1 h of ischemia, the abundance of the cap-initiation complex protein eIF4G is reduced by relocalization into TIAR protein complexes, triggering 4E-BP1 sequestration of eIF4E and disruption of cap-dependent mRNA initiation complexes. As the heart begins to fail at 6 h, proteolysis of eIF4G is observed, resulting in its depletion and accompanied by limited destruction of 4E-BP1 and eIF4E. eIF4G proteolysis and modest loss of 4E-BP1 are associated with caspase-3 activation and induction of cardiomyocyte apoptotic and necrotic death. Acute heart ischemia therefore downregulates cap-dependent translation through eIF4E sequestration triggered by eIF4G depletion.

High-efficiency gene transfer into skeletal muscle mediated by electric pulses.

Gene delivery to skeletal muscle is a promising strategy for the treatment of muscle disorders and for the systemic secretion of therapeutic proteins. However, present DNA delivery technologies have to be improved with regard to both the level of expression and interindividual variability. We report very efficient plasmid DNA transfer in muscle fibers by using square-wave electric pulses of low field strength (less than 300 V/cm) and of long duration (more than 1 ms). Contrary to the electropermeabilization-induced uptake of small molecules into muscle fibers, plasmid DNA has to be present in the tissue during the electric pulses, suggesting a direct effect of the electric field on DNA during electrotransfer. This i.m. electrotransfer method increases reporter and therapeutic gene expression by several orders of magnitude in various muscles in mouse, rat, rabbit, and monkey. Moreover, i.m. electrotransfer strongly decreases variability. Stability of expression was observed for at least 9 months. With a pCMV-FGF1 plasmid coding for fibroblast growth factor 1, this protein was immunodetected in the majority of muscle fibers subjected to the electric pulses. DNA electrotransfer in muscle may have broad applications in gene therapy and in physiological, pharmacological, and developmental studies.

The C-terminal helix of human apolipoprotein AII promotes the fusion of unilamellar liposomes and displaces apolipoprotein AI from high-density lipoproteins.

To assess the functional properties of apolipoprotein (apo) AII and to investigate the mechanism leading to the displacement of apo AI from native and reconstituted high-density lipoproteins (HDL and r-HDL) by apo AII, wild-type and variant apo AII peptides were synthesized. The wild-type peptides, residues 53-70 and 58-70, correspond to the C-terminal helix of apo AII and are predicted to insert at a tilted angle into a lipid bilayer. We demonstrate that both the apo AII-(53-70) peptide, and to a lesser extent the apo AII-(58-70) peptide are able to induce fusion of unilamellar lipid vesicles together with membrane leakage, and to displace apo AI from HDL and r-HDL. Two variants of the apo AII-(53-70)-wild-type (WT) peptide, designed either to be parallel to the water/lipid interface [apo AII-(53-70)-0 degrees] or to retain an oblique orientation [apo AII-(53-70)-30 degrees], were synthesized in order to test the influence of the obliquity on their fusogenic properties and ability to displace apo AI from HDL. The parallel variant did not bind lipids, due to its self-association properties. However, the apo AII-(53-70)-30 degrees variant was fusogenic and promoted the displacement of apo AI from HDL. Moreover, the extent of fusion of the apo AII-(53-70)-WT, apo AII-(58-70)-WT and apo AII-(53-70)-30 degrees peptides was related to the alpha-helical content of the lipid-bound peptides measured by infrared spectroscopy. Infrared measurements using polarized light also confirmed the oblique orientation of the helical component of the three peptides. In native and r-HDL, the tilted insertion of the C-terminal helix of apo AII resulting in a partial destabilization of the HDL external lipid layer might contribute to the displacement of apo AI by apo AII.

Apolipoprotein(a) yeast artificial chromosome transgenic rabbits. Lipoprotein(a) assembly with human and rabbit apolipoprotein B.

The in vivo analysis of lipoprotein(a) (Lp(a)), an independent atherosclerosis risk factor in humans, has been limited in part by its restricted distribution among mammals. Although transgenic mice have been created containing Lp(a), the relatively small size of the mouse has precluded some studies. To examine the properties of this molecule in a significantly larger mammal, we have used a 270-kilobase yeast artificial chromosome clone containing the human apolipoprotein(a) (apo(a)) gene and a 90-kilobase P1 phagemid clone containing the human apolipoprotein B (apoB) gene to create transgenic rabbits that express either or both transgenes. Expression of both transgenes was tissue specific and localized predominantly to the liver. Average apolipoprotein plasma levels in the rabbits were 2.5 mg/dl for apo(a) and 17.6 mg/dl for human apoB. In contrast to observations in apo(a) transgenic mice, we found that apo(a) plasma levels in the rabbits were stable throughout sexual maturity. Also, apo(a) formed a covalent association with the endogenous rabbit apoB albeit with a lower efficiency than its association with human apoB. The analysis of Lp(a) transgenic rabbits has provided new insights into apo(a) expression and Lp(a) assembly. In addition, these transgenic rabbits potentially will provide an improved experimental model for the in vivo analysis of Lp(a) and its role in promoting atherosclerosis and restenosis.

HDL3 binds to glycosylphosphatidylinositol-anchored proteins to activate signalling pathways.

Previous studies have indicated that in HepG2 cells HDL3-signalling involves glycosylphosphatidylinositol (GPI) anchored proteins. HDL3-binding to HepG2 cells was found to be enhanced by cellular preincubation with PI-PLC inhibitors and sensitive to a cellular preincubation with exogenous PI-PLC, suggesting that HDL3 binds directly on GPI-anchored proteins to initiate signaling. Moreover HDL3-binding was found to be partly inhibited by antibodies against the HDL-binding protein (AbHBP). HDL3, when binding to HepG2 cells, promoted the release in the culture medium of a 110 kDa protein that binds AbHBP, while a cellular preincubation with antibodies against the inositol-phosphoglycan (IPG) moiety of GPI-anchor (AbIPG), used to block lipolytic cleavage of the GPI-anchor, inhibits HDL3-induced release of the 110 kDa protein in the culture medium. In [3H]-PC prelabeled HepG2 cells, AbHBP were found to stimulate PC-hydrolysis and DAG generation within 5 min as did HDL3 stimulation. Cellular preincubation with AbIPG was found to inhibit only the HDL3-signal and not the AbHBP-signal, while a prior cellular pretreatment with PI-PLC from Bacillus cereus was found to inhibit the HDL3-and AbHBP-signal. Moreover cellular preincubation with AbHBP for 1 h at 37 degrees C was found to inhibit HDL3-signalling pathways. Our results suggest that in HepG2 cells a 110 kDa protein, which could be HBP, can be anchored to the membrane via GPI, and can function in HDL3-signalling pathways as binding sites.

Lipid-binding properties of synthetic peptide fragments of human apolipoprotein A-II.

Human apolipoprotein A-II (apo A-II) consists of three potential amphipathic helices of 17 residues each, which contribute to the lipid-binding properties of this apolipoprotein. The conformation and lipid-binding properties of these peptides, either as single-helix or as two-helix peptides, were investigated by turbidity, fluorescence, electron-microscopy and circular-dichroism measurements, and are compared in this article. The lipid affinity of shorter C-terminal segments of apo A-II was compared with those of the single-helix or two-helix peptides, to define the minimal peptide length required for stable complex formation. The properties of the apo-A-II-(13-48)-peptide were further compared with those of the same segment after deletion of the Ser31 and Pro32 residues, because the deleted apo-A-II-(13-30)-(33-48)-peptide, is predicted to form a long uninterrupted helix. The single helices of apo A-II could not form stable complexes with phospholipids, and the helix-turn-helix segment spanning residues 13-48 was not active either. The apo-A-II-(37-77)-peptide and the apo-A-II-(40-73)-peptide could form complexes with lipids, which appear as discoidal particles by negative-staining electron microscopy. The shortest C-terminal domain of apo A-II able to associate with lipids to form stable complexes was the apo-A-II-(40-73)-peptide, which consisted of the C-terminal helix, a beta-turn and part of the preceding helix. The shorter apo-A-II-(49-77)-peptide, and the helical apo-A-II-(13-30)-(33-48)-peptide, could also associate with phospholipids. The complexes formed were, however, less stable, as they dissociated outside the transition temperature range of the phospholipid. These data suggest that the C-terminal pair of helices of apo A-II, which is the most hydrophobic pair, is responsible for the lipid-binding properties of the entire protein. The N-terminal pair of helices of apo A-II at residues 13-48 does not associate tightly with lipids. The degree of internal similarity and the cooperativity between the helical segments of apo A-II is thus less pronounced than in apo A-I or apo A-IV. The N-terminal and C-terminal domains of apo A-II appear to behave as two distinct entities with regard to lipid-protein association.

Gene transfer of cytidine deaminase apoBEC-1 lowers lipoprotein(a) in transgenic mice and induces apolipoprotein B editing in rabbits.

Apolipoprotein (apo) B100 is an essential component of low-density lipoproteins (LDL) and lipoprotein(a) [Lp(a)]. In mammals, apoB can be edited post-transcriptionally to encode a truncated form of apoB (apoB48) that is unable to form either of these atherogenic lipoproteins. To study the effect of increasing hepatic apoB editing activity on formation of Lp(a), a recombinant adenovirus encoding rat apoBEC-1, the cytidine deaminase component of the apoB mRNA editing complex, was administered to human apoB/apo(a) transgenic mice. This resulted in expression of apoBEC-1 in hepatocytes of these mice, increased hepatic editing of human apoB mRNA, and decreased plasma levels of human apoB100 and Lp(a). The apoBEC-1 recombinant adenovirus was also administered to rabbits, an animal which, like humans, naturally lacks hepatic apoB editing. Expression of the exogenous apoBEC-1 in rabbit liver resulted in editing of up to 10% of apoB mRNA. Hepatic apoB editing was associated with lower LDL levels in these rabbits relative to those treated with a control adenovirus. However, LDL levels were elevated significantly in both animals as a result of adenovirus injection. These studies demonstrate that introduction of the cytidine deaminase apoBEC-1 is sufficient to induce hepatic apoB editing in an animal lacking this activity, and that induction of editing could serve as a novel approach for lowering plasma concentrations of the atherogenic lipoproteins Lp(a) and LDL.

Effects of lipoprotein(a) on the binding of plasminogen to fibrin and its activation by fibrin-bound tissue-type plasminogen activator.

Molecular assembly of plasminogen and tissue-type plasminogen activator (t-PA) at the surface of fibrin results in the generation of fibrin-bound plasmin and thereby in the dissolution of a clot. This mechanism is triggered by specific interactions of intra-chain surface lysine residues in fibrin with the kringle domains of plasminogen, and is further amplified via the interaction of plasminogen kringles with the carboxy-terminal lysine residues of fibrin that are exposed by plasmin cleavage. By virtue of its marked homology with plasminogen, apo(a), the specific apolipoprotein component of Lp(a), may bind to the lysine sites available for plasminogen on the surface of fibrin and thereby interfere with the fibrinolytic process. A sensitive solid-phase fibrin system, which allows the study of plasminogen activation at the plasma fibrin interface and makes feasible the analysis of products bound to fibrin, has been used to investigate the effects of Lp(a) on the binding of plasminogen and its activation by fibrin-bound t-PA. Plasma samples from human subjects with high levels of Lp(a) were studied. We have established that Lp(a) binds to the fibrin surface and thereby competes with plasminogen (Ki = 44 nM) so as to inhibit its activation. We have further shown that Lp(a) blocks specifically carboxy-terminal lysine residues on the surface of fibrin. To further explore the role of apo(a) on the Lp(a) fibrin interactions, we have performed ligand-binding studies using a recombinant form of apo(a) that contains 17 kringle 4-like units. We have shown that recombinant apo(a) binds specifically to fibrin (Kd = 26 +/- 8 nM, Bmax = 26 +/- 2 fmol/well) and that this binding increases upon treatment of the fibrin surface with plasmin (Kd = 8 +/- 4 nM, Bmax = 115 +/- 14 fmol/well). Altogether, our results indicate clearly that binding of native Lp(a) through this mechanism may impair clot lysis and may favor the accumulation of cholesterol in thrombi at sites of vascular injury.

Plasminogen binding by alpha 2-antiplasmin and histidine-rich glycoprotein does not inhibit plasminogen activation at the surface of fibrin.

alpha 2-antiplasmin (alpha 2-AP) exerts its inhibitory effect on fibrinolysis by rapidly inhibiting the plasmin evolved; in addition, it has been suggested that interference with the binding of plasminogen to fibrin, a function shared with histidine-rich glycoprotein (HRGP), may also be significant in inhibition of fibrinolysis. To elucidate if plasminogen binding by these two alpha 2-globulins may decrease the generation of plasmin by tissue-type plasminogen activator (t-PA) at the surface of fibrin, a system mimicking the fibrin/plasma interface was used. Attempts were made to differentiate the plasminogen binding from the plasmin inhibitory function of alpha 2-AP. The activation of human Glu-plasminogen (native plasminogen with NH2-terminal glutamic acid) by fibrin-bound t-PA was performed in a plasma environment using either normal plasma, alpha 2-AP- or HRGP-depleted plasmas supplemented with increasing amounts of the lacking protein, or in a reconstituted system with purified plasminogen and various concentrations of alpha 2-AP and HRGP. The activation of Glu-plasminogen in alpha 2-AP-depleted plasma containing a normal concentration of HRGP produced a time-dependent increase in the generation of plasmin. The addition of 1 microM-alpha 2-AP to this plasma prevented the formation of Lys-derivatives and produced a marked decrease (42%) in the number of plasminogen-binding sites. In contrast, the addition of 1.5 microM-HRGP to HRGP-depleted plasma containing a normal amount of alpha 2-AP produced only a modest (17%) decrease in the amount of plasmin(ogen) bound. Moreover, in a purified system the amount of plasminogen-binding sites and thereby of plasmin generated at the surface of fibrin in the presence of both alpha-2 globulins was similar to the amount generated in the presence of alpha 2-AP alone. These results indicate clearly that the formation of reversible complexes between plasminogen and alpha 2-AP does not interfere with the binding and activation of plasminogen at the fibrin surface. In contrast, the inhibition of plasmin by alpha 2-AP decreases importantly the number of plasminogen-binding sites (carboxyl-terminal lysines) and inhibits thereby the accelerated phase of fibrinolysis. It can be concluded that interference of the binding of plasminogen to fibrin by alpha 2-AP during plasminogen activation, does not play a significant role in inhibition of fibrinolysis, and that the plasminogen-binding effect of HRGP, if any, is obscured by the important inhibitory effect of alpha 2-AP.

Apolipoprotein(a) and plasminogen interactions with fibrin: a study with recombinant apolipoprotein(a) and isolated plasminogen fragments.

Lipoprotein(a) [Lp(a)], but not low-density lipoprotein (LDL), was previously shown to impair the generation of fibrin-bound plasmin [Rouy et al. (1991) Arterioscler. Thromb. 11, 629-638] by a mechanism involving binding of Lp(a) to fibrin. It was therefore suggested that the binding was mediated by apolipoprotein(a) [apo(a)], a glycoprotein absent from LDL which has a high degree of homology with plasminogen, the precursor of the fibrinolytic enzyme plasmin. Here we have evaluated this hypothesis by performing comparative fibrin binding studies using a recombinant form of apo(a) containing 17 copies of the apo(a) domain resembling kringle 4 of plasminogen, native Lp(a), and Glu-plasminogen (Glu1-Asn791). Attempts were also made to identify the kringle domains involved in such interactions using isolated elastase-derived plasminogen fragments. The binding experiments were performed using a well-characterized model of an intact and of a plasmin-digested fibrin surface as described by Fleury and Anglés-Cano [(1991) Biochemistry 30, 7630-7638]. Binding of r-apo(a) to the fibrin surfaces was of high affinity (Kd = 26 +/- 8.4 nM for intact fibrin and 7.7 +/- 4.6 nM for plasmin-degraded fibrin) and obeyed the Langmuir equation for adsorption at interfaces. The binding to both surfaces was inhibited by the lysine analogue AMCHA and was completely abolished upon treatment of the degraded surface with carboxypeptidase B, indicating that r-apo(a) binds to both the intrachain lysines of intact fibrin and the carboxy-terminal lysines of degraded fibrin. As expected from these results, both r-apo(a) and native Lp(a) inhibited the binding of Glu-plasminogen to the fibrin surfaces.(ABSTRACT TRUNCATED AT 250 WORDS)

Hedgehog lipoprotein(a) is a modulator of activation of plasminogen at the fibrin surface. An in vitro study.

Lipoprotein(a) (Lp[a]), a highly atherogenic lipoprotein particle, is the prominent apolipoprotein B-containing lipoprotein in the hedgehog (Laplaud PM et al, J Lipid Res 1988;29:1157-1170). In the present work, we studied the consequences of the structural homology between the specific Lp(a) glycoprotein, apoprotein(a), and plasminogen on the generation of plasmin by fibrin-bound tissue-type plasminogen activator. The activation of plasminogen was initiated by adding either native plasma or Lp(a)-free plasma supplemented with the equivalent of 0.25 mg/ml of either purified Lp(a) or albumin to a surface of fibrin prepared on micortitration plates and to which human tissue-type plasminogen activator was specifically bound. With the Lp(a)-free plasma, an increase in the binding and activation of plasminogen as a function of time was observed. In contrast, in the presence of Lp(a) (i.e., native plasma or the reconstituted system), a significant decrease in the binding of plasmin(ogen) (approximately 60%) was obtained. These data indicate that hedgehog Lp(a) interferes with the binding and activation of plasminogen at the fibrin surface and may thereby behave as a factor regulating the extent of fibrin deposition. These results support our previous data indicating that high levels of Lp(a) may have antifibrinolytic effects in humans (Rouy D et al, Arterioscler Thromb 1991;11:629-638), are in agreement with the observation that Lp(a) is a risk factor for atherosclerotic disease, and provide further support to the view of Lp(a) as a link between atherosclerosis and thrombosis.

Lipoprotein(a) impairs generation of plasmin by fibrin-bound tissue-type plasminogen activator. In vitro studies in a plasma milieu.

Apoprotein(a), (apo[a]), the specific antigen of lipoprotein(a) (Lp[a]), consists of structural domains (a serine protease unit, kringles 4 and 5) with marked homology to those of the corresponding domains in plasminogen. In this study, we have investigated the impact of this unique structural mimicry on the binding and activation of plasminogen by fibrin-bound tissue-type plasminogen activator at the plasma-fibrin interface. We found that the total amount of plasmin generated on the surface of fibrin was decreased in the presence of high concentrations of Lp(a): 197 +/- 65 fmol in plasmas with greater than 60 mg/dl Lp(a) versus 287 +/- 112 fmol in control plasmas. A similar effect was also apparent in the corresponding euglobulin fractions (554 +/- 169 fmol versus 754 +/- 310 fmol), the latter lacking the plasminogen-binding proteins alpha 2-antiplasmin and histidine-rich glycoprotein, but containing Lp(a). The difference between plasma samples was significant (p less than 0.05) as calculated from the percent decrease in plasmin generated from plasmas with high levels of Lp(a) relative to that generated in the paired controls with low Lp(a) levels. The involvement of Lp(a) was verified in a reconstituted system consisting of normal human plasma supplemented with 100 mg/dl of either purified Lp(a) or low density lipoprotein. Lp(a) produced a decrease of 30% in the generation of plasmin (180 fmol versus 255 fmol in plasma, and 485 fmol versus 705 fmol in the euglobulin fraction). Moreover, using a radiolabeled sheep antibody against human apo(a), we were able to demonstrate the binding of 40 fmol Lp(a) to fibrin during ongoing plasminogen activation. These results indicate that Lp(a) impairs the binding of plasminogen to fibrin and thereby decreases the generation of plasmin by occupying C-terminal lysine residues unveiled on the fibrin surface by plasmin degradation as recently reported (Circulation 1990;82[suppl III]:III-92). In consequence, impairment of fibrinolysis and accumulation of Lp(a) at sites of vascular injury may occur, factors that may be important in the development of atherosclerosis and associated thrombosis.

The mechanism of activation of plasminogen at the fibrin surface by tissue-type plasminogen activator in a plasma milieu in vitro. Role of alpha 2-antiplasmin.

The mechanism of activation of human Glu-plasminogen by fibrin-bound tissue-type plasminogen activator (t-PA) in a plasma environment or in a reconstituted system was characterized. A heterogeneous system was used, allowing the setting of experimental conditions as close as possible to the physiological fibrin/plasma interphase, and permitting the separate analysis of the products present in each of the phases as a function of time. The generation of plasmin was monitored both by spectrophotometric analysis and by radioisotopic analysis with a plasmin-selective chromogenic substrate and radiolabelled Glu-plasminogen respectively. Plasmin(ogen)-derived products were identified by SDS/PAGE followed by autoradiography and/or immunoblotting. When the activation was performed in a plasma environment, the products identified on the fibrin surface were Glu-plasmin (90%) and Glu-plasminogen (10%), whereas in the soluble phase only complexes between Glu-plasmin and its fast-acting inhibitor were detected. Identical results were obtained with a reconstituted system comprising solid-phase fibrin, t-PA, Glu-plasminogen and and alpha 2-antiplasmin. In contrast, when alpha 2-antiplasmin was omitted from the solution, Lys-plasmin was progressively generated on to the fibrin surface (30%) and released to the soluble phase. In the presence of alpha 2-antiplasmin or in plasma, the amount of active plasmin generated on the fibrin surface was lower than in the absence of the inhibitor: in a representative experiment the initial velocity of plasmin generation was 2.8 x 10(-3), 2.0 x 10(-3) and 1.8 x 10(-3) (delta A405/min) for 200 nM-plasminogen, 200 nM-plasminogen plus 100 nM-alpha 2-antiplasmin and native plasma respectively. Our results indicate that in plasma or in a reconstituted purified system containing plasminogen and alpha 2-antiplasmin at a ratio similar to that found in plasma (1) the activation pathway of native Glu-plasminogen proceeds directly to the formation of Glu-plasmin, (2) Lys-plasminogen is not an intermediate of the reaction and therefore (3) Lys-plasmin is not the final active product. However, in the absence of the inhibitor, Lys-plasmin and probably Lys-plasminogen, which is more readily activated to plasmin than is Glu-plasminogen, are generated as well.