Tumor necrosis factor-alpha in human arterial wall with atherosclerosis.

Tumor necrosis factor-alpha (TNF-alpha) was demonstrated in human normal and atherosclerotic aorta, iliac and femoral arteries by immunohistochemistry using monoclonal and polyclonal antibodies. TNF was present in the cells of the arterial wall and as granular and diffuse extracellular deposits in the connective tissue matrix. Quantitative determinations of TNF by ELISA showed mean values of 21.7 +/- 0.7 ng/100 mg total extracted protein in normal intima, 38.2 +/- 0.5 in intimal thickenings, 25.5 +/- 1.1 in fibrous plaques and 16.8 +/- 0.2 ng/100 mg total extracted protein in media. Intimal thickenings presented the highest amounts of TNF with a statistically significant difference when compared to normal intima (P less than 0.05) and media (P less than 0.01). TNF-alpha concentrations in arterial eluates were about 200 times higher than in the corresponding serum samples. Western blotting analysis confirmed TNF-alpha eluted from the arterial wall to be about 17 kDa similar to human recombinant TNF-alpha. TNF-alpha in human atherosclerotic wall could be actively involved in the inflammatory events associated with atherosclerosis.

Immunohistochemical localization of C5b-9, S-protein, C3d and apolipoprotein B in human arterial tissues with atherosclerosis.

The terminal C5b-9 neoantigens of the complement complex, S-protein (Vitronectin), C3c, C3d and apolipoprotein B were localized on 16 aortic fibrous plaques, 8 aortic intimal thickenings, 4 fatty streaks intimae, 12 coronary fibrous plaques, 3 coronary intimal thickenings, 6 femoral and 5 basilar fibrous plaques, using an indirect and double-staining immunoperoxidase method. The granular specific deposits were localized in the fibrous cap and deeper parts of the plaque or in the deeper intima and inner-third media of intimal thickenings and fatty streaks intimae, in relation to the degree of atherosclerotic involvement. The different localization of C5b-9 and S-protein demonstrated by the double-staining technique is more suggestive for the assembly of the complex into the arterial wall and not for its preformed passage from circulation. The relation of these immune deposits to the degree of fibrosis and necrosis and their presence from the initial stages through to the advanced lesions could ascribe a role to the complement system in atherosclerosis.

Interleukin-6 and interleukin-8 protein and gene expression in human arterial atherosclerotic wall.

Interleukin 6 (IL-6) and interleukin 8 (IL-8) are present in the human arterial atherosclerotic wall as cellular and extracellular deposits in the connective tissue matrix. Quantitative determinations of IL-6 by ELISA showed mean values of 27.6 +/- 3.3 ng/100 mg protein in normal intima, 37.3 +/- 2.1 ng/100 mg protein in fibrous plaque and 25.7 +/- 4.3 ng/100 mg total extracted protein in media. IL-8 levels were 3.5 +/- 0.6 ng/100 mg protein in normal intima, 11.3 +/- 2.1 ng/100 mg protein in fibrous plaque and 8.5 +/- 1.4 ng/100 mg total extracted protein in media. Fibrous plaques presented statistically significant higher levels of both IL-6 and IL-8. IL-6 and IL-8 gene transcripts were present in human iliac fibrous plaque and media prelevated at surgery indicating that a local production by the cells of the arterial wall participate to their accumulation. We also tested the role of complement activation in induction of IL-6 and IL-8 protein synthesis as well as the subsequent activation of endothelial cells. Only IL-8 was induced by complement activation and this may contribute to increased IL-8 levels found in the atherosclerotic wall. When exposed to terminal complement complexes, endothelial cells in culture also showed an increase of both DNA-synthesis and p70 S6 kinase activity indicating that complement is able to induce not only IL-8 synthesis but also cell activation. The presence of IL-6 and IL-8 in the arterial wall where complement activation also occurred, clearly show the involvement of inflammatory events in initiation and progression of atherosclerosis.

Immunoelectron-microscopic localization of the terminal C5b-9 complement complex in human atherosclerotic fibrous plaque.

The assembly of the terminal C5b-9 complement complex is a prime mechanism of complement-induced membrane damage followed by inflammatory response mediation and subsequent extensive tissue damage. In the assembly process the terminal complement components expose neoantigenic determinants which can be recognized by specific antibodies. Using such a specific antibody, affinity-purified rabbit IgG and by means of immunoelectron microscopy, the C5b-9 neoantigens were localized on the structures of the human fibrous plaque from 3 iliac and 3 femoral arteries obtained at surgery. The immunoelectron-dense deposits were localized on the cell debris, enmeshed in the connective tissue matrix, consisting of irregular particles that frequently had the shape and size of intracellular organelles or vesicles with concentric osmiophilic lamellae. No deposits could be found on the intact cells, on the connective tissue matrix or on cholesterol and lipid deposits. The presence of C5b-9 neoantigens deposits in the fibrous plaques frequently associated with other immune-related proteins indicates that complement activation has occurred in situ and could be related to the chronic progression of the atherosclerotic lesion.

Immunoelectron-microscopic localization of S-protein/vitronectin in human atherosclerotic wall.

S-protein/vitronectin is a multifunctional glycoprotein interacting with both complement activation and coagulation pathways. Its presence was investigated in 5 femoral and 5 iliac atherosclerotic human arteries, obtained at surgery, by immunoelectron microscopy using an affinity purified rabbit IgG specific for human S-protein/vitronectin. The immunoelectron dense specific deposits were found in both intimal thickenings and fibrous plaques in association with elastic fibers, collagen bundles and cell debris in the vicinity of elastin. Cell debris embedded in the collagen matrix were S-protein/vitronectin negative. S-protein/vitronectin was also absent on intact cells, lipid droplets and cholesterol clefts. All cell debris, however, was positive for C5b-9 deposits suggesting that complement activation had occurred at these sites with or without S-protein/vitronectin interaction. S-protein/vitronectin may play a role in the arterial wall defence by restricting the extent of complement activation.

Immunohistochemical localization of the terminal C5b-9 complement complex in human aortic fibrous plaque.

The terminal C5b-9 complex of the complement system was localized in 26 aortic, 3 iliac and 4 femoral human fibrous plaques using indirect immunofluorescence and immunoperoxidase. IgG, IgA, IgM, Clq, C3c, C4, C9 and fibrinogen were investigated simultaneously. All the fibrous plaques presented C5b-9 deposits appearing like thin threads in the fibrous cap and masses and spots in the amorphous areas. The extent and intensity were in agreement with the size of the fibrous plaques. The intimal thickenings presented less intense deposits which were absent in atherosclerosis-free samples. The C5b-9 deposits were frequently associated with immunoglobulins and complement components in the same areas. Whereas the demonstration of complement components reflected only a nonspecific trapping, the presence of assembled C5b-9 in the damaged tissues is more indicative of the involvement of complement activation in the tissue injury. The absence of C5b-9 in the atherosclerosis-free intima and its presence at lower intensity in the intimal thickenings than in the fibrous plaques suggest a pathogenic involvement in the chronic progression of the atherosclerotic lesion.

Immunoglobulins and complement components in human aortic atherosclerotic intima.

Concentration and preferential retention of immunoglobulins and complement components were studied in comparison with other plasma proteins in 42 human aortae with atherosclerosis. Saline and acid extracted IgG, IgA, IgM, C1q, C3c, C4, C9, C3A, C-reactive protein, alpha 1-antitrypsin, alpha 2-macroglobulin, albumin, transferrin and fibrinogen were quantitatively determined using the radial immunodiffusion. The fibrous plaques and their adjacent areas contained higher levels of each protein than intima with only fatty streaks. No significant differences were found between the fibrous plaques and their adjacent areas presenting intimal thickenings. Saline eluted IgG and IgA were significantly higher in the fibrous plaque intima than in intimal samples with fatty streaks and were the only proteins detected in the acid eluates. The complement components were present in all saline eluates, while C-reactive protein was found in 23 samples. Crossed immunoelectrophoretic studies showed the activation of saline C3 and C4. In 8 cases serum levels of the studied proteins were compared with their concentration in saline eluates obtained from intima and media. The immunoglobulins and complement components presented higher intima/serum and lower media/intima retention ratios than the other studied proteins suggesting their preferential retention in the intima. The presence of immune related proteins in the atherosclerotic intima and their preferential retention might be explained not only by an altered permeability but also in relation to their function.

Co-localization of terminal C5b-9 complement complexes and macrophages in human atherosclerotic arterial walls.

The co-localization of terminal C5b-9 complement complexes and macrophages was investigated in human arteries with atherosclerosis using a double-labeling immunohistochemical technique. Macrophages were found in all the atherosclerotic arteries, with the accumulation correlating positively with the degree of atherosclerosis. This accumulation was associated with an increase of C5b-9 deposits, as well as with an increase in the number of deposits containing both complement components and macrophages ('co-localization'). This co-localization was found to pertain both to intact macrophages and to macrophage remnants. These data suggest that C5b-9 complement complex might be formed on macrophages with subsequent promotion of inflammatory events and progression of the atherosclerotic lesions.

Presence of C5b-9 complement complex and S-protein in human myocardial areas with necrosis and sclerosis.

Myocardial fragments with acute infarction (10 cases), scars after chronic infarction (6 cases), areas with focal sclerosis and necrosis (8 cases) compared with normal myocardial areas (8 cases), were processed for indirect and double-labelling immunoperoxidase techniques to localize C5b-9 neoantigens, S-protein, C3d and apolipoprotein B. Granular masses of C5b-9 and C3d and diffuse areas of S-protein and apolipoprotein B were localized in the acute or chronically damaged areas but not in areas free of lesion. Double-labelling data revealed similarly damaged areas of localization for C5b-9 and S-protein, and for C3d and apolipoprotein B, respectively, on rather different than usual tissue structures. C5b-9 determination by ELISA from myocardial eluates revealed lower levels of neoantigens in normal areas (2.3 +/- 0.3 micrograms/g dried tissue), higher levels in areas with sclerosis (7.9 +/- 0.7 micrograms/g dried tissue) and the highest amounts in areas with acute infarction (11.1 +/- 1.2 micrograms/g dried tissue). The presence of C5b-9 neoantigens in damaged myocardial areas with a different localization than S-protein is suggestive of local complement activation.

Decay-accelerating factor regulates complement-mediated damage in the human atherosclerotic wall.

Decay-accelerating factor (DAF) is an intrinsic membrane inhibitor that regulates the activity of C3 and C5 convertases of the classical and alternative complement pathways. Using two monoclonal antibodies, IC6 and IA10, DAF was localized by immunohistochemistry using streptavidin-biotin-peroxidase complex or silver-intensified immunogold techniques in aortic, iliac and femoral samples obtained at surgery and autopsy from 32 patients. DAF was localized on the cells and in the connective tissue matrix of the arterial wall. Fibrous plaques and intimal thickenings presented larger amounts than fatty streaks, intimae and normal areas. By Western blotting analysis, DAF extracted from the arterial wall had a molecular weight of about 67 kDa. Using a double-labeling technique, DAF and C5b-9 complexes were co-localized on nucleated cells and on cell debris. The cells isolated after enzyme digestion of the arterial wall were tested for the protective role of DAF to complement-mediated damage. When DAF of the sensitized cells was blocked by monoclonal antibodies, complement-mediated cell lysis was enhanced from 10-15% to 60-70%. The effect of anti-DAF antibodies was dose-dependent. DAF blocking in the absence of antibodies used for sensitization led to a lysis under 10%. These data suggest a protective role of DAF against autologous complement activation, however insufficient to prevent complement activation in the human atherosclerotic wall.

Localization of the terminal C5b-9 complement complex in the human aortic atherosclerotic wall.

The terminal C5b-9 complement complex was investigated in 15 aortic, 2 femoral fibrous plaques and 5 fatty streaks aortic intimae using indirect immunofluorescence and immunoperoxidase. All the fibrous plaques presented C5b-9 deposit-like threads in the fibrous cap and masses in the amorphous areas of the plaque. The deposits were frequently associated with other immune-related proteins such as: IgG, IgA, IgM, Clq, C3c and C4 which were simultaneously investigated. Fatty streaks intimae presented no C5b-9 and complement component deposits. Whereas the demonstration of the complement components could merely reflect a non-specific trapping, the presence of assembled C5b-9 in the damaged tissue is more indicative of the involvement of complement activation in the progression of atherosclerotic lesions.

Characterization of circulating immune complexes in heart disease.

Circulating immune complexes (CIC) were characterized for the content in IgG, IgA, IgM, C3 and C4 in patients with heart disease. The levels of IgG, IgM and C4 in PEG-precipitates of patients' sera were significantly higher than those found in controls, and the precipitation profile was similar to that of rheumatoid arthritis patients. Differences were observed in the composition of CIC: IgM was highest in association with myocarditis, and C3 predominated in cases of valvular disease. Complex-bound C4 was significantly higher in patients with myocardial infarction which developed pericarditis either early or late in the evolution of the disorder. Antimyocardial antibodies could be detected in sera and in corresponding PEG-precipitates. The bulk of the data suggests that CIC might play a pathogenetic role in various heart diseases.

Cells carrying C5b-9 complement complexes in human atherosclerotic wall.

Fibrous plaques and intimal thickenings of 5 femoral and 5 iliac human arteries obtained at surgery were processed for indirect and double-labeling immunoelectron microscopy using an affinity purified rabbit IgG anti-C5b-9 neoantigen and the EBM 11 monoclonal antibody anti-human macrophages. The C5b-9 complexes were localized in intact cells, disintegrated cells and cell debris enmeshed in the connective tissue matrix. Some of the cell debris bearing C5b-9 deposits was found to be of macrophage origin. Endocyted or exocyted pieces of membrane with pore-forming C5b-9 complexes were also identified. Damage of cells by complement in atherosclerotic lesions may contribute to atherogenesis.

Isosorbide nitrates, nitroglycerin, and sodium nitroprusside induce vasodilation concomitantly with inhibition of carbonic anhydrase I in erythrocytes.

This study describes the relationship between nitroglycerin, isosorbide dinitrate, sodium nitroprusside, and carbonic anhydrase I, as well as the involvement of this carbonic anhydrase I in vasodilation. Two groups of coronary patients and a group of rabbits underwent treatment with the above-mentioned vasodilating drugs. The activity of red blood cell carbonic anhydrase was monitored and determined by the stopped-flow method. The results show that these drugs inhibit the activity of the isozyme in parallel to their vasodilating effect. The results of this study lead to the hypothesis that through the pH modifications induced by these vasodilators by the inhibition of carbonic anhydrase I, the isozyme may be involved in the regulation of vascular tonus.

Serum zinc and copper in patients with atherosclerosis and thromboangiitis obliterans.

The serum concentration of Zn and Cu were determined in 126 controls and 160 patients with atherosclerosis obliterans (AO) and 53 patients with thromboangiitis obliterans (TO). The concentration of serum Zn decreases with age both in controls and patients with AO in all the evolutive stages of the disease. In patients with TO, the concentration of serum Zn shows no correlation with age. The significantly lower values of serum Zn in controls and patients with AO over 61 yr than those below this age is attributed to the decreased Zn supply, especially to some individual cellular abnormalities. The values of serum Cu are higher in patients with AO and TO than in controls irrespective of the evolutive stage of the disease. This was related to cigarette smoking and inflammation. The Zn/Cu ratio in the serum is below 1 in patients with AO and TO and in controls over 61-yr-old. The pathogenetic role of this ratio in atherosclerosis and inflammation still remains a controversial question.

Serum zinc and copper in hyperlipoproteinemia.

Starting from experimental observations demonstrating that a high ratio of zinc to copper led to hypercholesterolemia in rats, serum Zn and Cu levels were measured by atomic absorption spectrophotometry in 65 normolipemic controls and in 100 subjects with various types of hyperlipoproteinemia (HLP). Serum Zn levels did not significantly differ from control values in any type of HLP. However, hyperlipoproteinemic patients with obvious clinical atherosclerosis displayed significantly lower serum-Zn concentration than hyperlipo-proteinemic subjects without clinical symptoms. On the other hand, when compared to control subjects, serum Cu levels were not found to be decreased, but rather increased, in hyperlipoproteinemic patients with or without atherosclerosis. As a result, the Zn∶Cu ratio appeared to be lower than normal in hyperlipoproteinemic patients with cardiovascular disease. It is conceivable that changes of these trace elements should be rather connected to vessel injury and associated disease than to HLP which, at least in humans, is not accompanied by a high Zn∶Cu ratio.

Bioavailability of nifedipine from different oral dosage forms in healthy volunteers.

The absolute and relative bioavailability of nifedipine (1) from different formulations administered as single oral doses in healthy volunteers was determined. Serum concentrations of 1 were measured by GC. The absolute bioavailability of 1 was 53% because of presystemic metabolism. The bioavailability of Adalat (Bayer) tablets, Nifedipina (Terapia) and Corinfar (VEB Arzneimittelwerk Dresden) sugar-coated tablets was 93%, 92% and 86% (respectively) as compared with Adalat capsules. The AUC were not significantly different. The Cmax and tmax values were different, indicating that the absorption of 1 showed differences in first-order rate constants of dissolution in the above mentioned order. Despite the differences among the formulations studied, each preparation may have its merits. In a multiple dose regimen of 20 mg 1 (Nifedipina, Terapia) t.i.d., minimal therapeutic drug levels were achieved and maintained during steady state, from the 1st d of treatment.

Serum calcium and salt restriction in the diet of patients with essential arterial hypertension.

Serum concentration of ionic calcium and the effect of dietary salt restriction on serum total calcium was studied in patients with essential arterial hypertension. The concentration of serum ionic calcium was significantly lower than in controls in all the evolutive stages of the disease (p less than 0.005; p less than 0.005; p less than 0.001). During salt restriction serum total calcium rises significantly (p less than 0.005) as compared with the values found under a salt containing diet. It was suggested that increased chronic ingestion of sodium or restriction of dietary salt produces alterations in Na-Ca exchanges across semipermeable membrane of the smooth muscle.

[Doppler ultrasonic examination in assessing the outcome of the therapeutic method in patients with arteriosclerosis obliterans of the lower extremities]. / Aportul examenului ultrasonic Doppler la aprecierea evolutiei metodei terapeutice la bolnavii cu ateroscleroza obliteranta a membreior inferioare.

The study included 62 patients with atherosclerosis obliterans of the lower extremities, of whom 47 in stage II, 5 in stage III and 10 in stage IV of the disease. In 22 patients were present single arterial obliteration, in 30 patients multiple, layered arterial obliterations, in 8 distal arterial obliterations and 2 a diffuse atherosclerosis of the lower extremities. Patients were examined in admission to the clinic, after the treatment performed at the clinic and at various intervals, during 2-4 years. The segmental arterial pressure, the pressure index and the ultrasonogram recorded with the Cardior electrocardiograph were followed-up. The arterial pressure and the pressure indices returned to normal values only after invasive procedures. The medical treatment can bring about the clinical regression and stabilization of the disease, as well as the improvement of the ultrasonogram. For the choice of therapeutic means, the pressure index is the best criterion, since it correlates with the severity of the disease. In the case of a pressure index 0.75, the medical treatment or the transluminal dilatation is preferred, whereas at an index of 0.7 the surgical revascularisation is considered necessary. Other ultrasonic parameters were also taken into account, such as the systolic passage time and the maximum velocity, which are less important in the attainment of the mentioned aims.(ABSTRACT TRUNCATED AT 250 WORDS)