TGF-beta1 downregulates CD36 and scavenger receptor A but upregulates LOX-1 in human macrophages.

Transforming growth factor-beta1 (TGF-beta1), a key cytokine for control of cell growth, extracellular matrix formation, and inflammation control, is secreted by many cells present in the arteriosclerotic plaque. Lipid accumulation in the vessel wall is regarded as an early step in atherogenesis and depends on uptake of modified low-density lipoprotein (LDL) by macrophages through scavenger receptors and their transformation into foam cells. Prominent members of the scavenger receptor family are the class A type I and II receptors (ScR-A), the class B receptor CD36, and the recently detected lectin-like oxidized LDL receptor-1 (LOX-1), which, unlike the native LDL receptor (LDL-R), are not feedback controlled. CD36 is responsible for >50% of modified LDL uptake into human monocyte-derived macrophages. We therefore studied whether TGF-beta1 influences expression and function of ScR-A, CD36, and LOX-1 in monocytes using RT-PCR and flow cytometry. Total uptake of oxidized LDL by monocytoid cells, reflecting the combined function of all scavenger receptors, was significantly reduced by TGF-beta1. At initially low picomolar concentrations, TGF-beta1 decreased CD36 mRNA and protein surface expression and ScR-A mRNA levels in the human monocytic cell line THP-1 and in freshly isolated and cultivated human monocytes, whereas LOX-1 mRNA was increased. Expression of LDL-R and beta-actin was not affected by TGF-beta1. In conclusion, depression of scavenger receptor function in monocytes by TGF-beta1 in low concentrations reduces foam cell formation. Together with matrix control by TGF-beta1, this may be important for atherogenesis and plaque stabilization.

Downregulation by tumor necrosis factor-alpha of monocyte CCR2 expression and monocyte chemotactic protein-1-induced transendothelial migration is antagonized by oxidized low-density lipoprotein: a potential mechanism of monocyte retention in atherosclerotic lesions.

The subintimal infiltration with monocytes is crucially involved in the development of complex atherosclerotic plaques. Monocyte chemotactic protein-1 (MCP-1) and its receptor CCR2 are important for monocyte extravasation and formation of atherosclerotic lesions. However, mechanisms of monocyte persistence in atherosclerotic plaques remain to be elucidated. Flow cytometric analysis revealed that monocytoid Mono Mac 6 cells that had transmigrated endothelium towards a MCP-1 gradient expressed higher levels of CCR2 than the non-migratory fraction, while input cells were intermediate, suggesting that high CCR2 levels are essential for transendothelial chemotaxis. Pretreatment of Mono Mac 6 cells or isolated human blood monocytes with the inflammatory cytokine tumor necrosis factor (TNF)-alpha dose- and time-dependently reduced MCP-1-induced transendothelial chemotaxis, which was inhibited by the CCR2 receptor antagonist 9-76 analog. This was paralleled by a decrease in CCR2 surface protein and mRNA expression. as assessed by flow cytometry and reverse transcription-polymerase chain reaction, inferring that inhibition of monocyte transmigration was due to downregulation of CCR2 to levels insufficient for chemotaxis. In contrast, treatment of monocytes with oxidized low-density protein (oxLDL) containing oxidized lipids, such as cholesteryl linoleate 13-hydroxide. but not with LDL, increased CCR2 protein and mRNA expression. Notably, oxLDL counteracted the TNF-alpha-mediated downregulation of CCR2 and CCR2-dependent transendothelial chemotaxis. Macrophage-colony-stimulating factor hardly affected CCR2 expression and function, suggesting that differentiation was not responsible for effects on CCR2. In conclusion, TNF-alpha impairs MCP-1-induced transendothelial migration of monocytes by downregulating CCR2 which appears critical for migration. Exposure to oxLDL antagonized the effects of TNF-alpha, and may thus contribute to monocyte retention and perpetuation of a chronic inflammatory reaction in unstable atherosclerotic lesions.

The expression of the lectin-like oxidized low-density lipoprotein receptor (LOX-1) on human vascular smooth muscle cells and monocytes and its down-regulation by lovastatin.

Accumulation of oxidatively modified low-density lipoprotein (oxLDL) in the vascular wall is a characteristic feature of atherosclerosis. oxLDL can be taken up into monocytes, smooth muscle cells, and endothelial cells by several known scavenger receptors such as scavenger receptor class A I and II, CD36, and CD68. A new lectin-like oxLDL receptor (LOX-1) was recently found in bovine and human endothelial cells. We studied whether LOX-1 is also expressed in other cells present in the atherosclerotic lesion and whether its expression can be modified. We found LOX-1 expression in human blood monocytes, umbilical smooth muscle and endothelial cells, and 3T3 fibroblasts. LOX-1 mRNA expression in monocytes could be significantly suppressed by lovastatin. Thus, LOX-1 expression is not restricted to endothelial cells and its down-regulation by HMG-CoA reductase inhibitors could contribute to the clinical benefits of these drugs.

Simultaneous solid phase extraction, derivatization, and gas chromatographic mass spectrometric quantification of thromboxane and prostacyclin metabolites, prostaglandins, and isoprostanes in urine.

The current analytical methods for the various prostanoids require a separate and extended sample workup, derivatization, and gas chromatographic/mass spectrometric detection of each compound. Therefore, we developed and validated a rapid method for the common purification, derivatization, and GC/MS determination of 11-dehydrothromboxane B2, 2,3-dinor-6-keto-PGF1a, PGF2A, PGE2, PGD2, and isoprostanes in urine. A single reversed-phase solid-phase extraction step and modified reaction conditions yielded excellent sample purification at high recoveries and efficient derivatization for all compounds in one vial. The method allows, for the first time, the simultaneous quantification of these index metabolites of systemic thromboxane and prostacyclin synthesis, renal prostaglandin formation, and nonenzymatic in vivo lipid peroxidation in a single GC/MS run with high sensitivity and precision.

Lovastatin reduces expression of the combined adhesion and scavenger receptor CD36 in human monocytic cells.

The thrombospondin and collagen receptor CD36 was recently found to function, also, as a dominating scavenger receptor for oxidized low-density lipoproteins (oxLDL). Thus, CD36 might be a key factor in monocyte adhesion and foam cell formation. We, therefore, studied CD36 expression in monocytic cells under conditions of cholesterol depletion and overload. Human monocytic U937 cells were cultured under control conditions and in the presence of lovastatin, native, and oxLDL. The expression of lipoprotein receptors was measured by quantitative reverse transcriptase polymerase chain reaction (RT-PCR) and fluorescence-activated cell sorting (FACS). In sharp contrast to the feedback-controlled ApoB100 specific receptor for native low-density lipoprotein (LDL-R), CD36 expression was significantly reduced by lovastatin in a dose-dependent manner, both at the RNA and protein level, resulting in decreased cellular oxLDL binding. The addition of mevalonate completely reversed lovastatin effects, whereas excess LDL was only partially effective. Similarly to native LDL, oxLDL reduced LDL-R transcription, but did not affect CD36 transcription. CD36 protein surface expression fell, however, due to internalization of CD36 loaded with oxLDL. In summary, monocytic expression of CD36, in contrast to the native LDL-R, is reduced by cholesterol synthesis inhibition and not by feedback inhibition from substrate overexposure. CD36 suppression is a new pharmacological action of lovastatin that may contribute to its clinical benefit by attenuating monocyte adhesion and foam cell formation, key steps in atherosclerosis.

Effects of dietary fish oil on ventricular premature complexes.

For ethical and practical reasons, in this study the antiarrhythmic potential of fish oil was evaluated in patients free from complex ventricular arrhythmias and severe heart failure. Although subjects without overt structural heart disease had ventricular arrhythmias that were not associated with an increased risk for sudden cardiac or coronary death, recent data suggest that frequent VPCs in patients similar to our study population may reflect subclinical cardiac disease amenable to the multiple beneficial actions of n-3 fatty acids. The potential and safety of fish oil as a treatment for more complex cardiac arrhythmias or arrhythmias in higher risk patients with more severe heart disease deserve further study.

N-3 but not N-6 fatty acids reduce the expression of the combined adhesion and scavenger receptor CD36 in human monocytic cells.

CD36, a multifunctional adhesion receptor e.g. for thrombospondin and collagen, as well as a scavenger receptor for oxidized low density lipoprotein, is expressed e.g. on platelets and monocytes. By this dual role it might be involved in early steps of atherosclerosis like the recruitment of monocytes and formation of foam cells. We therefore studied the effects of n-3 fatty acids on CD36 expression in human monocytic cells. Incorporation of eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) into cellular phospholipids resulted in a significant reduction of CD36 expression at the mRNA and protein level, whereas arachidonic acid (AA, C20: 4n-6) and linoleic acid (LA, C18:2n-6) tended to increase CD36 expression compared to the control. This specific down-regulation of CD36 by n-3 fatty acids in cells involved in the initiation and progression of atherogenesis and inflammation, represents a further mechanism that may contribute to the beneficial effects of n-3 polyunsaturated fatty acids (PUFA) in these disorders.

Evidence for sustained platelet activation in patients with early postinfarction angina.

Excretion of 2,3-dinor-thromboxane B2 released from activated platelets has been found elevated in patients with acute myocardial infarction and unstable angina. To investigate the role of thrombotic activity in postinfarction angina we measured urinary concentrations of 2,3-dinorthromboxane B2 in 20 patients over 10 days after myocardial infarction. Compared with 11 patients recovering pain-free, excretion of 2,3-dinorthromboxane B2 was found elevated in 9 patients who developed postinfarction angina. Accordingly, early postinfarction angina appears to be associated with ongoing thrombotic activity.

Rapid separation of the major phospholipid classes on a single aminopropyl cartridge.

A rapid method for the separation of the individual phospholipid classes phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI) by a single solid-phase extraction was developed. PC, PE, PS and PI were sequentially eluted from aminopropyl bonded silica with acetonitrile/n-propanol (2∶1, vol/vol), methanol, isopropanol/methanolic HCl (4∶1, vol/vol) and methanol/methanolic HCl (9∶1, vol/vol). Standard recoveries were over 95% for PC and PE and over 85% for PS and PI with undistorted fatty acid composition. The separation of complex lipid mixtures on aminopropyl minicolumns can be refined to the level of individual phospholipid classes.

A prospective study of the diagnostic value of urinary thromboxane in patients presenting with acute chest pain.

In chance single-case observations thromboxane excretion has been reported to increase several days prior to myocardial infarction. To test its frequency and potential diagnostic value we prospectively measured thromboxane excretion in 166 consecutive patients who had presented to the emergency unit with acute chest pain indicative of ischaemia. Thromboxane excretion at presentation was increased, sometimes dramatically, in 17 of 33 (52%) patients with unstable angina, in 42 of 73 (57%) patients with definite myocardial infarction, but in only two of 14 (14%) patients with stable angina. Nineteen of 29 patients undergoing early angiography had detectable intracoronary thrombi, and these patients excreted significantly more thromboxane than patients without thrombi. Ongoing platelet activation may be detected by increased thromboxane excretion in more than 50% of the patients presenting with unstable angina and myocardial infarction, particularly in those with intracoronary thrombi, but it is not a general phenomenon that can be used in diagnosis.

A critical evaluation of urinary immunoreactive thromboxane: feasibility of its determination as a potential vascular risk indicator.

Urinary immunoreactive thromboxane (irTXB2) has been found helpful in acute settings with altered renal, but also extrarenal thromboxane formation. As only trace amounts of systemically formed thromboxane are excreted unmetabolized, the nature of urinary irTXB2 was explored. The two most abundant metabolites of systemic thromboxane, 2,3-dinor-TXB2 and 11-dehydro-TXB2, crossreacted about 70% and less than 1%, respectively, with a widely used thromboxane antiserum. After solid-phase extraction of urine samples and separation on reversed-phase HPLC, the bulk of immunoreactivity always eluted as one peak shown to correspond to 2,3-dinor-TXB2. Much less was found in fractions where TXB2 eluted. Therefore, urines were read against calibration curves constructed with 2,3-dinor-TXB2. This direct estimation gave good recoveries for standard 2,3-dinor-TXB2 and correlated well, both in healthy controls and in patients at increased risk or with overt vascular disease, to values obtained after solid phase extraction, purification on reversed-phase HPLC and quantitation by either gas-chromatography mass-spectrometry or radioimmunoassay. Patients with multiple cardiovascular risk factors but free from detectable vascular disease excreted significantly more irTXB2 than age-matched controls with non-vascular conditions or normals. Therefore, urinary irTXB2 measured with this antiserum represents 2,3-dinor-TXB2, reflecting the systemic formation of TXB2. This simple approach is feasible for screening thromboxane formation in large series of patients. Its acumen in detecting the early development of vascular disease and its relation to established risk factors deserves large-scale prospective testing.

Superior antiplatelet action of alternate day pulsed dosing versus split dose administration of aspirin.

To explore the effect of timing on the antiplatelet action of aspirin, a constant mean amount of 40 mg aspirin/day was administered either as a split regimen of 20 mg twice daily, a single dose of 40 mg or a doubled dose of 80 mg every other day for 1 week each and compared to a current standard low dose regimen of 324 mg/day. Bleeding time, serum thromboxane, collagen-stimulated platelet aggregation and associated thromboxane formation and excretion of thromboxane and prostacyclin metabolites were measured both at peak and trough action of the drug. The inhibitory effects on platelet aggregation and associated thromboxane formation were significantly less marked with the split dose regimen, intermediate with the single dose of 40 mg aspirin/day and best with the alternate day doubled dose, but still inferior to the effects of 324 mg/day. Thromboxane excretion was suppressed by greater than 80% with all regimens. Prostacyclin metabolite excretion was similar for all 40 mg/day regimens with about 40% suppression at trough and 60% at peak drug action, respectively. Suppression was more pronounced after 324 mg/day. For best platelet inactivation at comparable sparing of prostacyclin formation, low doses of aspirin should be administered in pulsed rather than split regimens.

Biochemical evidence of platelet activation in patients with persistent unstable angina.

Thromboxane released from activated platelets and prostacyclin of the vessel wall may act as potent antagonistic modulators of platelet aggregability and coronary vascular tone. Therefore, urinary excretion of their major metabolites, 2,3-dinor-thromboxane B2 and 2,3-dinor-6-ketoprostaglandin F1 alpha, was studied in 16 patients presenting with prolonged angina at rest. The 10 patients whose condition did not improve under vigorous antianginal treatment within 48 hours exhibited higher thromboxane metabolite excretion than did the 6 patients who responded to therapy (2,208 +/- 1,542 versus 609 +/- 312 ng/g creatinine; p less than 0.001). Elevated values were also found in four of eight patients with sustained postinfarction angina. Enhanced thromboxane metabolite excretion was frequently associated with angiographic evidence of thrombus formation. When nine patients were restudied in a stable phase after 11 +/- 5 months, thromboxane metabolite excretion was consistently normal or high normal. Excretion of prostacyclin metabolites was not depressed in any patient but correlated weakly with thromboxane (r = 0.41). Thus, enhanced thromboxane production as an index of platelet activation may identify patients with active thrombus formation who could benefit most from platelet inhibitory treatment.

Effects on prostanoid formation and pharmacokinetics of dazmegrel (UK-38,485), a novel thromboxane synthase inhibitor, in man.

The pharmacokinetics of dazmegrel (UK-38,485), a novel selective thromboxane synthase inhibitor, and its effects on in vivo prostanoid formation were studied in a 2 weeks, multiple dose, placebo controlled, double blind trial in man. The drug was well tolerated. After dazmegrel 50-200 mg p.o. peak plasma levels of 0.7-3 mu/ml were reached within 1 hr. Elimination was of first order with a half life of 0.88 +/- 0.17 hr. Platelet count and bleeding time were unchanged by all regimes of dazmegrel used (100 and 200 mg b.i.d.; 50, 100 and 200 mg t.i.d.). Serum thromboxane (TXB2) was more than 95% suppressed one hour after all doses studied, but 200 mg t.i.d. were needed suppress circadian serum TXB2 profiles more than 90% at all times. Urinary excretion of 2,3-dinor-TXB2 (TXA2-M) fell by over 90%. An increase in the excretion of 2,3-dinor-6-keto-PGF1 alpha (PGI2-M), the major metabolite of prostacyclin, was largely transient and fell short of significance at all times. The ratio of TXA2-M to PGI2-M was lowered from about 5.0 to 0.2 and sustained throughout treatment. Dazmegrel selectively blocks in vivo and ex vivo TXA2 formation. Redirection of endoperoxides from total body TXA2 formation into prostacyclin formation is only minor under basal conditions.

[Prevention of thrombosis in the after-care of coronary dilatation and thrombolysis]. / Prävention der Thrombose in der Nachbehandlung von Koronardilatation und Thrombolyse.

An endothelial injury that leads to local thrombogenicity is produced during angioplasty and persists after successful thrombolysis. Thus the risk of thrombosis is augmented after these interventions. The high grade coronary stenosis remaining after lysis increases the shear forces, again stimulating thrombus formation. A higher recurrence rate after angioplasty is seen in the presence of a wall thrombus. Therefore antithrombotic therapy is of importance not only for prophylaxis of reocclusion but also for prevention of restenosis after angioplasty. During angioplasty reduction of wall thrombus formation by acetylsalicylic acid in addition to heparin could be shown, and is likely after lysis. A low dose regimen of acetylsalicylic acid appears to be as effective as high dose treatment and minimizes adverse gastrointestinal reactions. A reduction of thrombotic occlusions of about 50% seems possible. The platelet inhibiting effects of beta blockers an Ca antagonists are not of clinical importance. There is no perfect antiplatelet agent for prophylaxis of occlusion and restenosis, so a combination of drugs with different modes of action may be necessary.

Improved aortocoronary bypass patency by low-dose aspirin (100 mg daily). Effects on platelet aggregation and thromboxane formation.

Prevention of aortocoronary bypass occlusion by aspirin (ASA, 1 X 100 mg per day) was studied in a prospective double-blind trial of 83 patients. 60 (72%) were randomly allocated to ASA or placebo starting 24 h after operation. 90% of grafts in the ASA group and 68% in the placebo group were patent at four months. At least one anastomosis was occluded in 62% of the patients on placebo and in 27% of those on aspirin. Ventricular arrhythmias increased after surgery in more patients on placebo (12/18) than in patients on ASA (5/17). Platelet thromboxane formation on collagen tested before operation was significantly higher in patients in whom bypass occlusion developed (occlusion: 40 +/- 19, no occlusion: 25 +/- 13 ng/ml). A 100 mg dose of ASA per day effectively blocked platelet thromboxane formation and thromboxane-supported aggregation on collagen and was safe in the postoperative phase. No side effects were reported throughout the trial. The reduced toxicity with full efficacy favours a low and infrequent dosage of aspirin.