[Production of reactive oxygen species by monocyte-derived macrophages from the blood of healthy donors and patients with IHD].

Production of reactive oxygen species (ROS) by macrophages from blood monocytes of healthy donors (MP(N)) and patients with IHD (MP(IHD)) before, during, and after their incubation with low-density lipoprotein (LDL) isolated from the blood plasma of healthy donors (LDL(N)) and patients with a high cholesterol level (LDL(H)) was estimated by the method of luminol-dependent and stimulated by opsonized zymosan (OZ) or phorbol-12-myristate-13-acetate (PMA) chemiluminescence (CL). Intrinsic luminol-dependent, and zymosan- or PMA-stimulated chemiluminescence of MP(IHD) have exceeded the same types of chemiluminescence of MP(N) by factors of 1.4, 1.8, 2.7, and 1.6, respectively (p < 0.05-0.01). The effect of zymosan on MP(N) and MP(IHD) was stronger than that of TPA by factors of 4.3 and 3.2, respectively, but manifested itself 2.5-3.0 times slower. LDL(N) and LDL(H) incubated with MP(N) during 15-60 min increased ROS production by a factor of 1.4 and 2.5 respectively, but influenced ROS production by MP(IHD) (as estimated by luminol-dependent chemiluminescence). Effects LDL(N) and LDL(H) on MP(IHD) were not detected at all. Repeated increase in zymosan-stimulated CL of MP(N) was also observed after their 15-180 min preincubation with LDL(N) and LDL(H) which followed after taking out LDL, washing MP(N) and adding Hanks' solution with opsonized zymosan. This increase was also stronger after MP(N) incubation with LDL(H) than after MP(N) incubation with LDL(N), and no increase was observed in experiments with MP(IHD). Thus, the results obtained by a chemiluminescent method showed that fresh macrophages from the blood of patients with IHD had higher ROS production than macrophages from healthy donors. LDL(N) and LDL(H) could exhibit primary and secondary (after preincubation) stimulating effect on CL in MP(N); but had no effect on MP(IHD). An analysis of macrophage chemiluminescence is a sensitive test for evaluation the degree of macrophage's stimulation and it may be effectively used for the dynamic control for treatment effectiveness in clinics.

[LDL oxidation and uptake by monocyte-derived macrophages from blood of patients with ischemic heart disease].

The aim of this study was to test our hypothesis that monocyte-derived macrophages of patients with ischemic heart diseases (IHD, MPIHD) were prestimulated (primed) or stimulated cells whose capacity for LDL oxidation and uptake exceeded that ofmacrophages from healthy donors (MPN). Monocytes were obtained from the blood of 18 healthy donors and 25 IHD patients; plasma LDL--from 16 another group healthy donors (LDLN) and 15 patients with family hypercholesterolemia. Incubation of LDLN or LDLH with MPIHD or MPN was carried out under aerobic and hypoxic conditions. It was shown that incubation of LDLN or LDLH with MPIHD TBARS accumulation, LDL aggregation, apoB fragmentation were observed earlier and proceeded more actively than in the case of incubation with MPN. MPIHD (compared to MPN) more actively uptook LDLH and LDLN as accumulated greater amounts of total cholesterol (TCh) (by a factor of 1.8-2.1; p < 0.05-0.01), and their viability decreased to a markedly greater degree (p < 0.01). MPIHD and MPN also oxidized and took up LDLH with a higher intensity than LDLN, and their capacity for LDL oxidation and uptake increased, under hypoxic condition, compared to those under aerobic conditions. Thus, new experimental results provide direct evidence that macrophages of IHD patients are in vivo priming or stimulated cells and that this stimulation, especially in combination with hypercholesterolemic LDL and hypoxia, is a very strong risk factor that can predispose these patients to the onset or progression of atherosclerosis. Using MPIHD, it was created express-method for evaluation the degree of monocyte/macrophage stimulation in patients with IHD, selection of premedication medicine and new antiatherosclerotic and antiischemic drugs.

Effect of antioxidant probucol on cell-mediated LDL oxidation in vitro and in vivo.

We studied the effect of phenol antioxidant probucol on free radical oxidation of LDL isolated from blood plasma of healthy donors. Oxidation was induced by co-incubation of LDL with cultured peripheral blood monocyte-macrophages and human umbilical vein endothelial cells under conditions of ischemia-reperfusion. In addition, the effect of probucol therapy on oxidability of plasma LDL in CHD patients was examined. Probucol (0.1-10 microM) efficiently protected LDL from free radical oxidation in vitro and in vivo.

Tumor necrosis factor-alpha in low doses preactivates and activates macrophages by increasing their ability to produce reactive oxygen species and oxidize low-density lipoproteins: protective effect of antioxidants.

Tumor necrosis factor-alpha in low doses activated rat peritoneal macrophages and intensified production of reactive oxygen species (zymosan-depended chemiluminescence). Single or 2-fold incubation with tumor necrosis factor-a activated and preactivated human blood macrophages and promoted oxidative modification of low-density lipoproteins (increased their mobility in agarose gel). Antioxidants (potassium phenosan, probucol, and desferal) suppressed oxidative modification of low-density lipoproteins induced by nonactivated, preactivated, and activated macrophages. Our results show that antioxidants hold much promise for the prevention and therapy of atherosclerosis.

[The use of antioxidants and antihypoxants for decreasing of LDL oxidation by macrophages and endothelial cells in ischemia and reperfusion of vascular wall]. / Primenenie antioksidantov i antigipoksantov dlia snizheniia okisleniia LNP pod vliianiem makrofagov i éndotelial'nykh kletok v usloviiakh ishemii i reperfuzii sosudistoi stenki.

Oxidative modification of LDL is a key factor in pathogenesis of atheroslerosis. In this work the effects of antioxidants (K-phenosan, probucol, and desferal) and antihypoxants (succinic acid, hypoxen, and deltaran) on the macrophage- and endothelial cell-mediated oxidation of LDL was studied. Electrophoretic mobility of LDL, the content of lipid peroxide products (TBARS and diene conjugates, DC) and cell viability were used as the indexes of LDL oxidation. The effectiveness of antioxidants as inhibitors of LDL oxidation decreased in the following order: desferal > probucol > K-phenosan, and antihypoxant ability was decreased in line: deltaran >> succinic acid (effect only in dose 40 mg/ml) >> hypoxane (no effect). The effect antioxidants on protection of cell viability (MP and EC) during ischemia reduced in the same order. The effectiveness of antihypoxant protection of MP viability, decreased in the following order: succinic acid > hypoxen >> deltaran. Macrophages added to 24 h ischemized EC + LDL in early reperfusion period decreased LDL EPM. This may apparently be attributed to selective uptake of oxidized LDL by MP.

[Comparative evaluation of the cytotoxic effect of hydrogen peroxide and tumor necrosis factor alpha on nonischemic and ischemic endothelial cells]. / Sravnitel'naia otsenka tsitotoksicheskogo éffekta perekisi vodoroda i faktora nekroza opukholi al'fa na neishemizirovannye i ishemizirovannye éndotelial'nye kletki.

We studied cytotoxic effects (CTE) induced in confluent cultures of human umbilical vein endothelial cells (HUVEC) by initiators of free-radical reactions (FRR): H2O2 (10(-6)-10(-9) M), recombinant human tumor necrosis factor-[symbol; see text] (TNF-alpha, 0.05-100 ng/ml), and a combination of TNF-alpha with low-density lipoproteins (LDL, 100 microgram/ml). HUVEC were incubated with these substances for 6 or 24 h in parallel tests performed under aerobic (CO2-incubator) and ischemic conditions (a mixture of 95% N2 + 5% CO2 in RPMI-1640 medium containing no substrate additives, growth factor or protein). HUVEC viability was determined by counting cells adherent to the bottom of wells after 24 h of reincubation under aerobic conditions in the growth medium (Plating Efficiency Index). The data showed that: 1) CTE of these compounds were dose-dependent (H2O2 and TNF-alpha) and time-dependent (TNF-alpha); 2) CTE of FRR initiators and CTE of ischemia were synergistic, that is, their combination produced a greater decrease HUVEC viability than any substance examined or ischemia alone; 3) CTE of TNF-alpha observed in experiments in substrate-deficient, protein-free medium was considerably stronger than in the growth medium; 4) a combination of TNF-a and LDL caused a stronger CTE on HUVEC than either factor alone, and this synergism was more pronounced during incubation under ischemic conditions. Thus, the data indicate that FRR initiators and TNF-alpha + LDL particularly increase the severity of ischemic injuries of EC and therefore they can be factors which in hypercholesterolemic patiens predispose vascular wall to atherosclerosis.

[The evaluation of the possibility of using prodigiozan for isolating extracts that stimulate proliferative processes in the resected liver]. / Otsenka vozmozhnosti ispol'zovaniia prodigiozana dlia vydeleniia ékstraktov, stimuliruiushchikh proliferativnye protsessy v rezetsirovannoi pecheni.

The experiments were performed on 126 white male rats. The inclusion of 3H-thymidine in nuclear DNA of the liver was studied on h 24 and 48 after 70% resection of the liver versus prodigiosan injection or combination of prodigiosan with 70% resection of the liver. Liver extracts stimulating proliferation (ESP) obtained under the above schedules were studied on the model of 30% liver resection. It is shown that prodigiosan (0.25 mg/kg) injected to intact rats and rats with 70% resected liver initiated ESP production promoting the inclusion of 3H-thymidine in nuclear DNA of the liver after 30% resection of the liver.

[The effect of the antioxidant ionol as a liposome component on the function of the isolated and ischemic heart]. / Vliianie antioksidanta ionola v sostav liposom na funktsiiu izolirovannogo i ishemizirovannogo serdtsa.

The anti-ischemic and toxic effects of different doses of the antioxidant ionol (butyl hydroxytoluene, BHT) introduced into the Krebs-Henseleit medium composed of monolayer liposomes of egg phospholipids before or 30 minutes after total ischemia of the heart were studied on a model of perfusion of isolated Wistar rat heart by the method of Langendorff-Fallen. It has been demonstrated that ionol, after its addition to the perfusate in the preischemic period, exerts an anti-ischemic effect in concentrations of 10(-6), 3 x 10(-6) and 10(-5) M; in the postischemic period, in concentrations of 3 x 10(-6) and 10(-5); the protective effect of ionol in the postischemic period is less pronounced. In higher doses (3 x 10(-5) and 10(-4) M) ionol produces a toxic action which is more remarkable and is less reversible in respect to the ischemized than to the nonischemized heart, and is realized rather through the dysfunction of heart muscle relaxation. The conclusion is drawn that there is a possibility of using ionol in doses of 10(-6) to 10(-5) M as a constituent of liposomes for addition to perfusion, conservant and cardioplegic solutions as an anti-ischemic remedy.

[Changes in EEG and higher nervous activity of rats during cerebral anti-ischemic protection by acelysin]. / Izmenenie EEG i VID krys pri ispol'zovanii dlia protivoishemicheskoi zashchity golovnogo mozga atselizina.

The water-soluble aspirin (acelysin) has been used as an anti-ischaemic protector when injected in the dose of 150 mg/kg 30 min before ischaemia. The EEG has been registered during the whole period of experiment and the total EEG power index has been calculated. The higher nervous activity has been evaluated during analysis of rat's abilities for elaboration of conditional reflex of an active escape reaction in Y-labyrinth. The results have demonstrated the complete rehabilitation and restoration of brain functional activity 8 days after endurance of brain ischaemia under protection of acelisin.

[Anti-ischemic protection of the brain using water-soluble form of aspirin-acelisin]. / Protivoishemicheskaia zashchita golovnogo mozga s pomoshch'iu vodorastvorimoi formy aspirina-atselizina.

The domestic water-soluble aspirin (acelisin) has been used as an anti-ischemic brain protector. The total brain ischemia has been implemented in accordance with an original technique for 17 to 35 min. The doses of acelisin from 25 to 250 mg/kg have been tested during experiments. The infusion of solutions has been carried out before ischemia, 15 min before reperfusion and just after the beginning of reperfusion. The functional status and survival of rats have been evaluated during a week. The best result has been reached with 150 mg/kg acelisin injected 30 min before ischaemia. A positive effect was reported when acelisin was used in early postischaemic period.

[Anti-ischemic action of a 1-hydroxy-2,2,6,6-tetramethylpiperidine derivative from a series of nitroxyl bioantioxidants]. / Protivoishemicheskoe deistvie 1-gidroksi-proizvodnogo 2,2,6,6-tetrametilpiperidina iz riada nitroksil'nykh bioantioksidantov.

The experiments have been performed on 216 Wistar rats to examine anti-ischaemic action of the 1-hydroxy-2,2,6,6-tetramethyl-piperidine derivative (I), whose antioxidant properties were, earlier shown for model systems. Introduction of I (10(-4) M) into the perfusion medium and the subsequent storage (37 degrees C) of isolated liver was shown to decrease the accumulation of lipid peroxidation products (MDA). Compound I administrated (5 x 10(-6)-10(-5) M) in perfuse medium of isolated (Langendorff method) and ischemized (30 min, 37 degrees C) heart improves contractile function (Pmax) and decreases end-diastolic pressure at postischaemic period. In vivo injection of I increases (12 mg/kg, i.p.) the number of rats survival after sublethal time (2.5 h) of liver total ischaemia, increase (35 mg/kg, i.p.) the number of rats survival and improves parameters of heart function after ischaemic shock (6 h ischaemia and reperfusion of limbs). The analog of I, corresponding amine, possessing no antioxidant properties also fails to exhibit any anti-ischaemic effect.

[Is xanthine oxidase a universal source of superoxide radicals in ischemic and reperfusion lesions?]. / Iavliaetsia li ksantinoksidaza universal'nym istochnikom superoksidnykhradikalov pri ishemicheskom i reperfuzionnom povrezhdeniiakh?

While studying xanthine-xanthine oxidase system it was found, that a considerable accumulation of xanthine and uric acid occurred whereas xanthine dehydrogenase did not transfer in xanthine oxidase during 2 hours of total rat liver ischemia. These data make it possible to reject the generally accepted hypothesis of xanthine oxidase key role in free radical mechanism of ischemia damage.

[Membranes of subcellular organelles as the source of superoxide radicals in liver ischemia]. / Membrany subkletochnykh organell kak istochnik superoksidnykh radikalov pri ishemii pecheni.

O2-generation rate (Vo2-) in microsomal, mitochondrial and nuclei liver membranes was measured by ESR method, by accumulation of stable nitroxide radicals. These Vo2- values were compared with Cu, ZnSOD and MnSOD activities after 2 hours ischemia and 24 hours reoxygenation. O2- radicals generated by electron transfer chains are concluded to damage mainly during the ischemia, but not the reoxygenation.