Urokinase plasminogen activator system in humans with stable coronary artery disease.

1. The present study compares plasma urokinase plasminogen activator (uPA) peptide levels, plasma plasminogen inhibitor (PAI-1) activity and urokinase receptors (uPAR) on peripheral blood monocytes of patients with stable coronary artery disease (SCAD) and healthy volunteers. 2. Urokinase plasminogen activator levels were analysed by ELISA and PAI-1 activity was determined by a plasmin generation method using the chromogenic substrate S2390. Relative uPAR numbers and the adhesion molecules CD11b/CD18 on peripheral blood monocytes were estimated using specific antibodies and flow cytometry. 3. Patients with SCAD were found to have higher plasma uPA peptide levels than age-matched healthy subjects (10.40 +/- 0.99 vs 8.25 +/- 0.53 pmol/L, respectively; P < 0.05). 4. Plasma PAI-1 activity was also higher in patients with SCAD than in healthy subjects (13.6 +/- 2.5 vs 5.2 +/- 1.0 IU/mL, respectively; P < 0.05). 5. Relative uPAR and CD11b/CD18 adhesion molecules were similar on peripheral blood monocytes of patients with SCAD and in healthy subjects. 6. The data indicate a pattern of expression/activity of uPA and PAI-1 in patients with SCAD suggestive of an impaired fibrinolytic ability.

Autoantibodies to gangliosides in sera of atherosclerotic patients.

Using ELISA we studied the levels and clinical correlation of serum antibodies against gangliosides and 5-hydroxytryptamine (5-HT) in patients with atherosclerosis and clinical manifestations of cardiovascular disease. A range of 70-80% of the patients showed higher titers of anti-GM3(L) and anti-5HT as compared to normal serum. The anti-GM3(L) antibodies appeared to be directed mainly against GM3 present in platelets and were much less reactive against GM3 isolated from the aorta. We concluded that the antigens responsible for the elevated anti-GM3(L) and anti 5-HT levels in atherosclerotic sera are released by vessel-wall activated platelets. These results provide further evidence of on-going autoimmune processes in atherosclerosis. The content of total sialic (TS) and lipid-bound sialic acid (LBS) was measured in sera of patients with IHD and of similar numbers of healthy donors. In the patient groups the average TS and LBS concentration was about 25% higher than in the control group. These changes appeared to be associated with higher degrees of protein sialylation and larger amounts of LDL in the patient sera than in those of healthy controls.

Diagnostic value of immune cholesterol as a marker for atherosclerosis.

BACKGROUND: The serum of patients with coronary atherosclerosis contains circulating immune complexes including low-density lipoproteins (LDLs). We have developed a technique for the evaluation of LDL content in circulating immune complexes by measuring total cholesterol levels in polyethylene glycol precipitates (immune cholesterol). In the present study, the value of immune cholesterol in the diagnosis of atherosclerosis was compared with that of other laboratory parameters, such as total cholesterol levels, triglycerides, LDL cholesterol, high-density lipoprotein cholesterol, lipoprotein(a), and apolipoproteins B and A-1. METHODS: Immune cholesterol and the other parameters were determined in blood samples from 200 patients with documented coronary and extracoronary atherosclerosis. Coronary atherosclerosis was assessed by coronary angiography; stenoses in the aortic arch and branches and in lower-limb arteries were evaluated by angiography and ultrasonography. RESULTS: Only immune cholesterol and the ratio of apolipoprotein B to apolipoprotein A-1 correlated significantly with the severity of coronary atherosclerosis. The accuracy of the diagnosis of coronary atherosclerosis by immune cholesterol was 78%, considerably higher than that of other laboratory parameters. Use of a combined parameter consisting of immune cholesterol, LDL cholesterol, and the patient's age increased the diagnostic accuracy to 81%. A high level of immune cholesterol is characteristic not only of coronary atherosclerosis but also of extracoronary atherosclerosis. The sensitivity, specificity and accuracy of the diagnosis of extracoronary atherosclerosis were even higher than those for coronary atherosclerosis. CONCLUSION: Immune cholesterol may be employed as a novel marker in the diagnosis of advanced atherosclerosis.

Extracorporeal immunoadsorption for the specific removal of lipoprotein (a) (Lp(a) apheresis): preliminary clinical data.

The extracorporeal procedure for the specific removal of lipoprotein (a) (Lp(a)) from human plasma--Lp(a) apheresis--was applied to the treatment of three patients with coronary artery disease documented by angiography. Their initial lipid levels were as follows: total cholesterol, 210-230 mg/dl; low-density lipoprotein (LDL) cholesterol, 140-160 mg/dl; Lp(a), 90-120 mg/dl. The patients underwent a total of 168 procedures without significant side effects. Lp(a) apheresis reduced the Lp(a) level by removing up to 88% of Lp(a). Other plasma compounds, including LDL and plasminogen, remained practically unchanged. Lp(a) apheresis appears to be a unique, effective and specific method for lowering the Lp(a) level. Additional trials are needed to evaluate the clinical effect of this treatment.

Platelets in patients with homozygous familial hypercholesterolemia are sensitive to Ca(2+)-mobilizing activity of low density lipoproteins.

While some data suggest that Ca(2+)-mobilizing effects of low density lipoprotein (LDL) in platelets are mediated by a specific membrane receptor, the data about the nature of this lipoprotein-binding site are contradictory. This work was performed in order to assess possible involvement of apolipoprotein (apo) B,E receptor, present in most cell types. To answer the question we compared effects of LDL in normal platelets and those obtained from patients with homozygous familial hypercholesterolemia (HFH), characterized by absence of functional apo B,E receptors. We have found that in accordance with previous results LDL induced instant reversible elevation of free cytoplasmic calcium concentration ([Ca2+]i) in fura-2-loaded platelets. The effect was observed both in healthy and HFH groups. Neither half-maximal effective concentrations nor maximal effects of LDL differed significantly between two groups. Ca(2+)-mobilizing effects of lipoproteins were potentiated about 4-fold by epinephrine and completely blocked by prostaglandin E1 both in platelets of healthy and HFH subjects. The similarity of lipoprotein effects in control and HFH platelets is evidence that apo B,E receptor does not mediate the Ca(2+)-mobilizing activity of LDL in this cell type.

Use of cultured atherosclerotic cells for investigation of antiatherosclerotic effects of anipamil and other calcium antagonists.

Calcium antagonists have been shown to exhibit an antiatherosclerotic action in primary cultures of human aortic atherosclerotic cells by causing a reduction in intracellular lipid content, proliferative activity and synthesis of the extracellular matrix. Verapamil and nifedipine exhibited the highest efficacy in this respect. The new calcium antagonist, anipamil (racemate and enantiomers), has been tested in cell cultures. At 10(-6) M and higher concentrations, anipamil and its enantiomers produced considerable decrease in intracellular content of cholesteryl esters, triglycerides and free cholesterol, suppressed cell proliferation and inhibited synthesis of the extracellular matrix. The efficacy of anipamil (enantiomers and racemate) was similar to that of verapamil and greater than that of nifedipine. Plasma obtained from patients after administration of 80 mg verapamil or 20 mg nifedipine significantly lowered the cholesterol content of cultured cells. Blood plasma of most atherosclerotic patients possesses atherogenic properties, i.e., it is able to increase the cholesterol content in cultured cells. Plasma atherogenicity seen in cultures decreased considerably or even disappeared after both nifedipine and verapamil administration. After 28 days of nifedipine therapy, plasma atherogenicity was lower compared with the initial value at the beginning of the treatment. These observations suggest that control of plasma atherogenicity after drug administration may provide an additional tool for optimisation of direct antiatherogenic and antiatherosclerotic therapy.

LDL- and agonist-induced Ca(2+)-mobilization in platelets of healthy subjects and in patients with familial hyperlipoproteinemia type II.

LDL induced a reversible increase in [Ca2+]i in platelets of healthy subjects and FH-patients. In both groups the effects of LDL were potentiated 2.3-fold by epinephrine. [Ca2+]i increases, induced by LDL, ADP and PAF were more prominent in platelets of FH-patients. This may explain the platelet hyperaggregability in such conditions.

Correlation between cholesterol content in circulating immune complexes and atherogenic properties of CHD patients' serum manifested in cell culture.

Blood serum of most patients with coronary heart disease (CHD) caused a 2-5-fold increase in the total cholesterol content of smooth muscle cells cultured from unaffected human aortic intima, i.e. possessed an atherogenic potential manifested in culture. Removal of immunoglobulins G and M from an atherogenic serum brought about a fall in its atherogenic potential. The serum deficient in immunoglobulins A retained its ability to induce the cholesterol accumulation in cells. Treatment of the CHD patients' serum with 2.5% polyethylene glycol 6000 removed the circulating immune complexes. The serum subjected to this treatment lost its atherogenicity, i.e. failed to increase the cholesterol content in cultured cells. Incubation of smooth muscle cells derived from human aortic intima with circulating immune complexes isolated from an atherogenic patients' serum caused a 1.5-3-fold rise in the intracellular cholesterol. Blood sera of most (89%) CHD patients was characterized by a high cholesterol content in circulating immune complexes. More than 75% of healthy subjects and patients without stenosis of coronary arteries had low level of cholesterol in immune complexes. Blood sera atherogenicity manifested in culture directly correlated with the cholesterol level of circulating immune complexes (r = 0.90). These findings suggest that the atherogenicity of CHD patients blood serum is due to cholesterol-containing immune complexes.

Antiatherosclerotic effects of calcium antagonists. Study in human aortic cell culture.

To investigate the effects of calcium antagonists on atherosclerotic cellular indices, [3H]thymidine incorporation and intracellular cholesterol content, primary culture of cells isolated from subendothelial intima of human atherosclerotic aorta was used. Among tested drugs were: verapamil, nifedipine, diltiazem, papaverin, nicardipine, D-600, cinnarizine, PN 200 110 and PY 108 068. Verapamil proved to be the most effective. It significantly reduced the total intracellular cholesterol and sharply decreased the incorporation of [3H]thymidine. With respective efficacy verapamil was followed by nifedipine and PY 108 068. Neither beta-blocker (metoprolol) nor nitrate (nitroglycerin) modified antiatherosclerotic effects of calcium antagonist (nifedipine). Calcium agonist Bay K 8644 which facilitates the penetration of calcium into cells caused the accumulation of intracellular cholesterol and stimulated cell proliferation. Simultaneous addition of nifedipine and Bay 8644 led to the inhibition of the agonist's atherogenic effect. Thus, facilitation of calcium influx into cells causes atherosclerotic alterations in the arterial cells; atherogenic calcium effects are inhibited by calcium channel blockers. Furthermore, based on the results of application of blood plasma from patients treated with calcium antagonists or beta-blocker to primary cultures of atherosclerotic cells, it can be assumed that calcium antagonists affect an anti-atherosclerotic and beta-blockers an atherogenic action.

Peritoneal macrophages: a model for detecting atherogenic potential in patients' blood serum.

Human and mouse peritoneal macrophages cultured in the presence of the blood serum of patients with documented coronary heart disease showed a 2- to 3-fold rise in levels of intracellular cholesterol ester and a 1.5- to 2-fold increase in those of free cholesterol and triglycerides. This effect was observed in 83% of cases, whereas the serum of healthy subjects induced the accumulation of lipids in macrophages only in 28% of cases. These data accord with previously published observations obtained on smooth muscle cells of human aortic intima. A direct correlation was found between the accumulation of cholesterol in macrophages and in cultured smooth muscle cells of human aortic intima. The accumulation of lipids in macrophages was dose dependent and increased with time. It is assumed that a culture of peritoneal macrophages may serve as a model for identifying an atherogenic potential of patients' blood serum.

Blood serum atherogenicity associated with coronary atherosclerosis. Evidence for nonlipid factor providing atherogenicity of low-density lipoproteins and an approach to its elimination.

To reveal the presence of atherogenic potential in the blood serum obtained from patients with angiographically assessed coronary atherosclerosis we used primary cultures of subendothelial cells isolated by collagenase from unaffected human aortic intima. Earlier, we have demonstrated that such cultures are made up mostly of typical and modified smooth muscle cells. Within 24 hours of cultivation with a 40% sera of patients suffering from coronary atherosclerosis, the total intracellular cholesterol level increased twofold to fivefold. Cultivation with the sera of healthy subjects had no effect on the intracellular cholesterol level. The sera of patients were separated by ultracentrifugation into two fractions: total lipoprotein fraction containing the main classes of lipoproteins and a lipoprotein-deficient fraction. The former, but not the lipoprotein-deficient fraction, was characterized by atherogenicity (i.e., the ability to induce the accumulation of intracellular cholesterol). Lipoproteins of the patients' serum were separated into main classes: low density lipoproteins (LDL), very low density lipoproteins (VLDL), and high density lipoproteins (HDL2 and HDL3). An atherogenic component of the serum capable of stimulating the deposition of intracellular cholesterol was represented by LDL and, in one case, by VLDL, but not by other classes of lipoproteins. LDL and other lipoproteins isolated from the blood serum of healthy subjects failed to raise the cholesterol content in cultured cells; that is, they were nonatherogenic.(ABSTRACT TRUNCATED AT 250 WORDS)

Cardiovascular drugs and atherosclerosis: effects of calcium antagonists, beta-blockers, and nitrates on atherosclerotic characteristics of human aortic cells.

Primary cell culture derived from atherosclerotic plaque of human aorta was used to assess direct effects of calcium antagonists, beta-blockers, and nitrates on vessel wall cells. Within 24 h, calcium antagonists (verapamil, nifedipine, darodipine, isradipine, diltiazem, etc.) reduced the cholesterol level in cultured cells. Furthermore, these agents decreased the incorporation of [3H]thymidine. Thus, the calcium antagonists manifested direct antiatherosclerotic action in culture by normalizing major manifestations of atherosclerosis at the cellular level. On the other hand, beta-blockers (propranolol, alprenolol, metoprolol, atenolol, pindolol, and timolol) caused a 1.5- to twofold rise in cholesterol level of cultured cells and stimulated their proliferation. Nitrates (nitroglycerin, isosorbide dinitrate, and nitroprusside) had no effect on atherosclerotic characteristics. Within 2-4 h after a single dose of oral administration of beta-blocker (propranolol), patients' blood plasma turned atherogenic, i.e., its addition to culture-induced cholesterol accumulation and stimulated proliferation. At the same time, blood plasma of patients who received calcium antagonists (verapamil and nifedipine) acquired antiatherosclerotic properties manifested in its ability to lower the intracellular cholesterol level and inhibit proliferative activity of cultured cells. These findings allow the assumption that not only in vitro, but in vivo as well, calcium antagonists and beta-blockers are antiatherosclerotic and atherogenic drugs, respectively.

Atherogenicity of blood serum from patients with coronary heart disease.

The sera from 62 of 68 patients with coronary heart disease (CHD) caused a two to five fold elevation in the intracellular cholesterol in primary cultures of subendothelial cells derived from grossly normal intima of human aorta. The sera from 33 of 42 healthy subjects did not show atherogenic properties in culture. Atherogenic potential correlated directly with the serum apolipoprotein-B-apolipoprotein A1 ratio, but not with the level of total cholesterol, high-density-lipoprotein cholesterol, apo-B, or apo-A1. The sera from patients with CHD also facilitated deposition of lipids in the medial smooth muscle cells of human aorta and mononuclear blood cells, though to a lesser degree. They had no such effect on endothelial cells of human aorta and umbilical vein, or human embryo fibroblasts.