Hypoxia induces differential expression of the integrin receptors alpha(vbeta3) and alpha(vbeta5) in cultured human endothelial cells.

The integrins alpha(vbeta3) and alpha(vbeta5) have been implicated in playing a key role in the process of angiogenesis. In this study, we examined the effects of hypoxia, an important stimulus of angiogenesis, on the differential expression of the integrin subunits beta(3) and beta(5). beta(3) and beta(5) messenger RNA (mRNA), protein levels, and alpha(v)beta(3) function were measured in human umbilical vein endothelial cells (HUVECs) cultured under normoxic and hypoxic (1% O(2)) conditions. Cells exposed to hypoxic conditions for up to 72 h showed gradually increased mRNA levels of alpha(V) and beta(3), peaking at 24 h, in comparison with cells cultured under normoxic conditions. However, beta(5) mRNA levels, under the same hypoxic conditions, remained at a constant level. Results from Western blot analysis of HUVECs, cultured under hypoxic conditions, paralleled those of the Northern analysis with an increased expression in alpha(v)beta(3) protein levels, measured by blotting with LM609, evident by 24 h. alpha(v)beta(5) protein levels, measured by blotting with P1F6, did not change for up to 72 h. HUVECs cultured under hypoxic conditions for 72 h showed increased attachment to fibrinogen, an alpha(v)beta(3) mediated process. These results indicate that hypoxia can increase expression of alpha(v)beta(3) in HUVECs, and that hypoxic regulation of alpha(v)beta(3) may be an important regulator of angiogenesis.

A monoclonal antibody that recognizes the GPIIb/IIIa antagonist DMP 728. Reversal of the effects of DMP 728 on platelet aggregation and bleeding time in the dog.

Since hemorrhagic events represent a major safety concern associated with the use of new antithrombotic therapies such as glycoprotein (GP) IIb/IIIa receptor blockade, we evaluated the ability of a monoclonal antibody recognizing DMP 728 (cyclic [D-2-aminobutyryl-N2-methyl-L-argininyl-glycyl-L-aspartyl-3- aminomethyl-benzoic acid] methanesulfonic acid salt), a potent GPIIb/IIIa receptor antagonist, to reverse the pharmacological actions of DMP 728 in the dog. DC11 was chosen for in vivo evaluation based on its ability to inhibit the binding of [3H]DMP 728 to activated platelets and to attenuate the inhibition of ADP-induced aggregation on platelet-rich plasma ex vivo by DMP 728. After anesthesia mongrel dogs were given DMP 728 (20 micrograms/kg body wt IV) infused into the femoral vein, bleeding times were determined using a Simplate device from incisions on the backside of the tongue, and platelet aggregation was determined ex vivo. Nearly complete inhibition of platelet aggregation was observed for the dogs treated with DMP 728 (20 ug/kg IV) for up to 210 minutes, and bleeding times were prolonged > 15 minutes for 2 hours and remained elevated for more than 4 hours. DC11 (0.2 or 1.0 mg/kg body wt IV) given to dogs 10 minutes after DMP 728 resulted in 50% attenuation of the effect of DMP 728 on aggregation at 3 hours. Approximately 34% inhibition of the DMP 728-mediated bleeding time was achieved at 1 hour with the 0.2 mg/kg dose, whereas approximately 50% inhibition of the bleeding time was observed for the 1 mg/kg dose at 1 hour.(ABSTRACT TRUNCATED AT 250 WORDS)

Recombinant plasminogen activator inhibitor-1 protects platelets against the inhibitory effects of plasmin.

Plasmin-induced degradation of platelet glycoprotein Ib (GPIb), the von Willebrand factor (vWF) receptor, has been implicated as a mechanism contributing to the development of platelet dysfunction following cardiopulmonary bypass (CPB). The goal of this study was to assess whether biologically active recombinant plasminogen activator inhibitor-1 (rPAI-1), could antagonize the inhibitory effects of plasmin on GPIb. GPIb function, as evaluated by measuring vWF-dependent, ristocetin-induced platelet agglutination in human platelet rich plasma (PRP) was significantly impaired following incubation with plasmin (60 +/- 14% inhibition, p < 0.01). Inclusion of rPAI-1 (10 micrograms/ml) in the PRP antagonized this plasmin effect, restoring agglutination to 92 +/- 8% of the control value (p < 0.01). The effect of rPAI-1 on the enzymatic activity of plasmin was further evaluated in an amidolytic assay with the plasmin substrate S2251 where an apparent second order rate constant of plasmin inhibition by rPAI-1 of 9.4 x 10(4) M-1 S-1 was determined. Our results suggest that rPAI-1, by inhibiting both tissue plasminogen activator-induced plasmin generation and plasmin activity directly, may have clinical value for improving platelet function during and after CPB.

Purification and characterization of active and latent forms of recombinant plasminogen activator inhibitor 1 produced in Escherichia coli.

Plasminogen activator inhibitor 1 (PAI-1), the principal physiological inhibitor of tissue plasminogen activator (tPA), is a protein of 379 amino acids and belongs to the SERPIN family of serine protease inhibitors. We have previously described methods to express [Sisk et al. (1990) Gene 96, 305-309] and purify [Reilly et al. (1990) J. Biol. Chem. 265, 9570-9574] a highly active form of the protein in substantial amounts, from Escherichia coli. Further analyses of this material showed the presence of small but significant amounts of latent rPAI-1. The present paper describes for the first time purification of latent and active forms of rPAI-1 from a single preparation, as well as the functional and structural characteristics of the two forms. Latent rPAI-1, which has properties similar to the latent forms described by other groups, was separated from active rPAI-1 by high-resolution ion-exchange chromatography or by affinity chromatography using immobilized anhydrotrypsin. It had low intrinsic activity (< 5% of active rPAI-1) and was partially reactivated by guanidine hydrochloride treatment or by incubation with vitronectin. Conversion of the active rPAI-1 to the latent form was influenced by temperature and additives including sucrose, EDTA, and arginine. Active and latent rPAI-1 did not show any obvious differences in their primary structures and displayed remarkably similar secondary structures as determined by circular dichroism spectral analyses. However, they did exhibit differences in tryptophan fluorescence, suggesting tertiary structural differences between the two forms.

Do.OH scavenger secondary radicals protect by competing with oxygen for cellular target sites?

Recently, using Chinese hamster V79 cells, we found no relationship between the level of protection and the overall rate for .OH removal [Ewing and Walton, Radiat. Res. 126, 187-197 (1991)]. We offered several possible interpretations for this observation, including that the scavengers may actually have multiple ways to protect, ways that would occur in addition to, or instead of, simple .OH removal. With bacterial spores, we had noted that protection occurs only with those .OH scavengers that are able to react and form secondary, reducing radicals (alpha-hydroxy radicals, RCOH), and we suggested that protection might occur if these radicals reduced cellular radical sites in competition with (damaging) reactions of O2. We have now tested that hypothesis with four .OH scavengers (DMSO, ethanol, glycerol, and methanol), and Chinese hamster V79 cells, irradiated while equilibrated with 0.9% O2 and 100% O2; our recent experiments with these scavengers in air provide data for a third O2 concentration. If these scavengers protect in vitro mammalian cells by forming secondary reducing radicals which compete with O2 for damaged cellular sites, we expect that when we reduce the O2 concentration, we will concomitantly reduce the scavenger concentrations needed for protection. If the proposed competition occurs, we expect the scavenger concentrations for 50% maximum effect to occur in the ratio of the three O2 concentrations used approximately 1:20:100. We found no evidence for such a competition as the mechanism of protection for these four .OH scavengers.

Radiation protection of in vitro mammalian cells: effects of hydroxyl radical scavengers on the slopes and shoulders of survival curves.

We have tested several chemical compounds, characterized and widely used as hydroxyl radical (.OH) scavengers, for their effects on the radiation sensitivity of Chinese hamster V79 cells irradiated in air or nitrogen. Our purpose is to reexamine the proposed relationship between the level of protection and the rates at which the scavengers react with .OH. We found that the additives can have two apparently independent effects on the shape of survival curves: a reduction in sensitivity (i.e., "protection," a decrease in the value of k) and an increase in the size of the shoulder of the survival curve (an increase in the value of Dq). We measured intracellular scavenger concentrations, and, using these values in our analysis, we found that neither of the two effects is correlated with the rates at which the scavengers react with .OH. Although these results could mean that .OH do not cause lethal damage, the interpretation we believe most probably correct is that these scavengers protect in multiple ways. The protection would occur in addition to or instead of simple .OH removal.

A spectroscopic study of the conformations of active and latent forms of recombinant plasminogen activator inhibitor-1.

Recombinant plasminogen activator inhibitor-1 (rPAI-1) purified from Escherichia coli, like its natural counterpart, can exist in either active or latent form. To elucidate the structural basis for these two forms, both active and latent rPAI-1 have been studied using ultra-violet (UV), circular dichroism (CD), and fluorescence spectroscopy. The secondary structures determined by CD show no significant differences and indicate that both the forms are predominantly alpha helical and random. The UV spectra are also very similar with absorption maxima around 278 nm. The structures of the two forms were further characterized by measuring tryptophan fluorescence emissions and their quenching with ionic (iodide) and neutral (acrylamide) quenchers. These data indicate clear differences in the tertiary structures of the two forms with the latent form being more compact and folded in comparison with the active form.

The relationship between the anoxic sensitivity and the extent of sensitization by nitrous oxide.

Nitrous oxide reacts during irradiation to increase the yield of .OH, a radical many believe to be a major cause of lethality. Logically, one would expect N2O to be a radiation sensitizer. In some instances it is, while in others it is not. In some cases we can explain why N2O fails to sensitize; factors such as dose rate, cell concentration, buffer composition and ionic strength all influence when N2O will sensitize and, if it sensitizes, by what magnitude. Based on the results presented here with multiple strains of procaryotic and eucaryotic cells, we believe the anoxic sensitivity is another critical factor that governs whether N2O will sensitize. Our data, with data from the literature, show a relationship between the anoxic sensitivity and the N2O enhancement ratio. N2O does not sensitize in vitro unless the anoxic sensitivity (inactivation constant, k) is less than approximately 0.2 daGy-1.

Purification and characterization of recombinant plasminogen activator inhibitor-1 from Escherichia coli.

A recombinant form of plasminogen activator inhibitor-1 (rPAI-1) has been purified from lysates of pCE1200, a bacterial expression vector containing the full length PAI-1 gene, by utilizing sequential anion exchange and cation exchange chromatography on Q-Sepharose and S-Sepharose columns. Approximately 140 mg of rPAI-1, estimated at 98% purity on the basis of analytical high performance liquid chromatography, could be obtained from 200 g wet weight of cells. The purified protein exhibited a single Coomassie Blue-stainable band at the region of Mr = 42,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and an NH2-terminal amino acid sequence consistent with the expected translation product of the pCE1200 PAI-1 insert. The rPAI-1 rapidly inhibited single- and two-chain tissue plasminogen activators, as well as urokinase, with apparent second order rate constants in the range of 2-5 x 10(7) M-1 s-1. A specific activity measurement of 250,000 units/mg was calculated for the rPAI-1 based on its ability to inhibit the enzymatic activity of a single-chain tissue plasminogen activator. Stability studies showed that the activity of the rPAI-1 was very stable when stored at temperatures of 25 degrees C or lower, but decayed within hours when stored at 37 degrees C. Sodium dodecyl sulfate treatment, which partially activates the latent form of natural PAI-1, inactivated rPAI-1. These results show that the purified rPAI-1 produced from pCE1200 displays many of the properties associated with the biologically active form of natural PAI-1.

Radiosensitizer conjugation to the carcinoma 19-9 monoclonal antibody.

Misonidazole was covalently conjugated (3-68 mol drug/mol antibody) to 19-9 monoclonal antibody directed against a colorectal carcinoma tumor-associated antigen as a method for targeting radiosensitizing agents. This attachment was accomplished by the mixed anhydride method using the hemisuccinate derivative of misonidazole. Evaluation of conjugates in vitro shows a loss of antibody binding activity with increasing loading levels; however, significant binding activity is retained even at relatively high sensitizer/antibody ratios. This observation was consistent in three binding assays: a competitive radioimmunoassay; an enzyme immunoassay; and an affinity column assay. From these studies, it was concluded that the optimal loading factor for misonidazole-antibody conjugates, when it is used for immunochemotherapy lies between 8 and 15. In vitro release studies indicated that conjugates are hydrolytically stable (t1/2 = 4 days) under physiological conditions.

Radiation sensitization by oxygen of in vitro mammalian cells: is .O-2 involved?

Oxygen is a potent sensitizer of cells exposed to ionizing radiation, and, although the exact chemical mechanisms are not fully understood, some evidence suggests that this sensitization may involve the formation of superoxide anion radicals (.O-2) [F. Lavelle, A. M. Michelson, and L. Dimitrijevic, Biochem. Biophys. Res. Commun. 55, 350-357 (1973); A. Petkau and W. S. Chelack, Int. J. Radiat. Biol. 26, 421-426 (1974); L. W. Oberley, A. L. Lindgren, S. A. Baker, and R. H. Stevens, Radiat. Res. 68, 320-328 (1976)] To test this hypothesis, we compared the sensitivity of Chinese hamster V79 cells irradiated in O2/N2 and O2/N2O gas mixtures with and without the addition of other radical scavenging agents. In these tests, although oxygen was present, be blocked the radiation-induced reactions of O2 which produce .O-2. We found that the total amount of biological damage depends simply on the concentration of O2 that is present; the overall sensitivity is not reduced when .O-2 cannot be formed. Thus radiation sensitization by O2--at least of this cell line--does not require the formation of superoxide anion radicals.