Secretome of apoptotic peripheral blood cells (APOSEC) attenuates microvascular obstruction in a porcine closed chest reperfused acute myocardial infarction model: role of platelet aggregation and vasodilation.

Although epicardial blood flow can be restored by an early intervention in most cases, a lack of adequate reperfusion at the microvascular level is often a limiting prognostic factor of acute myocardial infarction (AMI). Our group has recently found that paracrine factors secreted from apoptotic peripheral blood mononuclear cells (APOSEC) attenuate the extent of myocardial injury. The aim of this study was to determine the influence of APOSEC on microvascular obstruction (MVO) in a porcine AMI model. A single dose of APOSEC was intravenously injected in a closed chest reperfused infarction model. MVO was determined by magnetic resonance imaging and cardiac catheterization. Role of platelet function and vasodilation were monitored by means of ELISA, flow cytometry, aggregometry, western blot and myographic experiments in vitro and in vivo. Treatment of AMI with APOSEC resulted in a significant reduction of MVO. Platelet activation markers were reduced in plasma samples obtained during AMI, suggesting an anti-aggregatory capacity of APOSEC. This finding was confirmed by in vitro tests showing that activation and aggregation of both porcine and human platelets were significantly impaired by co-incubation with APOSEC, paralleled by vasodilator-stimulated phosphoprotein (VASP)-mediated inhibition of platelets. In addition, APOSEC evidenced a significant vasodilatory capacity on coronary arteries via p-eNOS and iNOS activation. Our data give first evidence that APOSEC reduces the extent of MVO during AMI, and suggest that modulation of platelet activation and vasodilation in the initial phase after myocardial infarction contributes to the improved long-term outcome in APOSEC treated animals.

Efficient phagocytosis of periodontopathogens by neutrophils requires plasma factors, platelets and TLR2.

BACKGROUND: Periodontitis represents a chronic infection of supportive dental tissues by distinct gram-negative bacteria. It is characterized by chronic and local inflammation as well as transient bacteremia with frequently occurring infections at distant sites. OBJECTIVES: The present work aimed to clarify the role of platelets and plasma factors in neutrophil interactions with the periodontopathogens A. actinomycetemcomitans and P. gingivalis. METHODS: Phagocytosis, cell-cell interactions and activation of platelets and neutrophils in response to periodontopathogens were analyzed by flow cytometry, confocal microscopy and bacteria survival assay. Plasma factors, platelet signaling pathways and receptors involved in platelet-neutrophil-bacteria interactions were determined. The role of platelet and neutrophil TLR2 in phagocytosis was further evaluated in a murine TLR2 knockout model. RESULTS: In the presence of plasma neutrophil-mediated clearance of periodontopathogens is doubled due to opsonisation of bacteria. Platelets, which become activated by periodontopathogens, further enhance clearance of bacteria by 20%, via direct interaction with neutrophils. Plasma factors (e.g. CD14) are required for platelet activation, which is mainly TLR2 dependent and results in PI3K/Akt activation. In a murine TLR2 knockout model we prove that platelet TLR2 is important for formation of platelet-neutrophil aggregates and enhanced phagocytosis of periodontopathogens. In contrast, neutrophil TLR2 is not involved in platelet-neutrophil aggregate formation but is required for efficient phagocytosis. CONCLUSIONS: These data indicate that efficient elimination of periodontopathogens by neutrophils involves a complex interplay of plasma factors as well as platelets and requires functional TLR2. By enhancing neutrophil activation platelets contribute to immune defense but may also foster inflammation.

Intravenous immunoglobulins induce CD32-mediated platelet aggregation in vitro.

BACKGROUND: Intravenous immunoglobulins (IVIg) and cytomegalovirus immunoglobulins (CMVIg) are currently finding increased acceptance in clinical states of high immune activity and in transplant recipients. A rare side-effect of their application is intravascular thrombosis, which is thought to be related to pre-existing hyperviscosity. In a previous study we have shown that rabbit antithymocyte globulin causes platelet aggregation in vitro via the Fc IgG receptor (CD32). OBJECTIVES: To investigate if IVIg and CMVIg have the potential to cause CD32-dependent platelet aggregation. METHODS: The influence of CMVIg or IVIg on platelets pre-incubated with or without monoclonal antibody AT10 was studied in an aggregometer. Expression of platelet surface activation marker CD62P was determined by fluorescence-activated cell sorting analysis and presence of soluble CD40L (sCD40L) was evaluated by enzyme-linked immunosorbent assay. All in vitro experiments were performed using platelet concentrates from the blood bank, at therapeutic concentrations of immunoglobulins. Results Incubation of platelets with CMVIg and IVIg markedly induced platelet aggregation, and increased expression of CD62P and secretion of sCD40L. The capacity of CMVIg and IVIg to induce platelet aggregation was completely abrogated by adding the blocking antibody AT10 directed against the low-affinity Fc IgG receptor (CD32). CONCLUSIONS: Our results suggest that CMVIg and IVIg solutions with activating Fc domains are able to bind CD32 on platelets and cause platelet aggregation in vitro. These results indicate a mechanism by which in vivo intravascular thrombosis may be explained and suggest caution with concomitant use of packed platelets and IVIg in autoimmune diseases in the clinical setting.

Effect of non-selective, non-steroidal anti-inflammatory drugs and cyclo-oxygenase-2 selective inhibitors on the PFA-100 closure time.

The place of cyclo-oxygenase (COX)-2 selective non-steroidal anti-inflammatory drugs (NSAIDs) in the peri-operative period remains under discussion. Due to the absence of COX-2 in platelets, the risk of bleeding in patients who use selective NSAIDs is thought to be decreased. We studied the influence of aspirin, diclofenac, lornoxicam and rofecoxib on the in vitro bleeding time using the platelet function analyser (PFA-100). The PFA-100 simulates the process of platelet adhesion and aggregation after vascular injury in vitro. Measurements in 43 volunteers were performed at three time points: before, 3 h, and 12 h after oral ingestion of one of the randomly assigned study medications. Aspirin, diclofenac and lornoxicam had a significant effect on the in vitro closure time, while rofecoxib did not show this effect. This supports the use of COX-2 selective drugs in the peri-operative period to minimise the risk of bleeding.

Inhibition of inducible nitric oxide synthesis by the herbal preparation Padma 28 in macrophage cell line.

Padma 28 is a mixture of herbs used in traditional Tibetan medicine with anti-inflammatory activities. We investigated the effects of Padma 28 on nitric oxide (NO) production by the inducible nitric oxide synthase (iNOS) in lipopolysaccharide stimulated mouse macrophages (RAW 264.7). Padma 28 (0-900 microg/mL) induced a concentration dependent inhibition of inducible nitric oxide synthesis. iNOS protein expression showed a concentration dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of Padma 28. Padma 28 decreased iNOS mRNA levels as shown by RT-PCR. Aqueous extracts from costi amari radix (costus root, the dried root of Saussurea lappa) and the outer cover of myrobalani fructus (the dried fruit of Terminalia chebula), constituents of the complex herb preparation Padma 28, were found to inhibit inducible nitric oxide synthesis by decreasing iNOS protein and iNOS mRNA levels. The inhibition of inducible nitric oxide synthesis might contribute to the anti-inflammatory activities of Padma 28.

Hypochlorite modified LDL are a stronger agonist for platelets than copper oxidized LDL.

Experimental low density lipoprotein (LDL) oxidation is usually performed using trace copper, although the in vivo relevance of this method has been called into question. Such LDL augment adenosine 5'-diphosphate (ADP) induced platelet aggregation, presumably by the action of lipid derived compounds. In striking contrast, we find that LDL oxidized to a comparable extent by hypochlorite, an in vivo occurring oxidant, reveal themselves to be potent promoters of platelet aggregation. Interestingly, hypochlorite modified LDL seem to mediate their influence on human platelets by means of the modified apolipoprotein B-100 (apoB) moiety. Also, the finding that hypochlorite modified albumin is able to trigger platelet aggregation suggests an essential role for hypochlorite modified protein(s) in the process of platelet activation.

Inhibition of inducible nitric oxide synthesis by oxidized lipoprotein(a) in a murine macrophage cell line.

Increased plasma levels of human lipoprotein(a) (Lp(a)) are highly correlated with the development of atherosclerotic lesions. During our study, we investigated the effects of native and hypochlorite oxidized lipoprotein(a) (ox-Lp(a)) on nitric oxide production by the inducible nitric oxide synthase (iNOS) in lipopolysaccharide/interferon stimulated mouse macrophages (J774A.1). Ox-Lp(a) (0-2 microg/ml) induces a dose dependent inhibition of inducible nitric oxide synthesis. iNOS protein expression showed a dose dependent reduction as revealed by immunoblotting when cells were incubated with increasing amounts of ox-Lp(a). Ox-Lp(a) decreases iNOS mRNA synthesis as shown by reverse transcription-polymerase chain reaction. Ox-Lp(a) induced iNOS inhibition might contribute to the development of atherosclerotic lesions by reducing the anti-atherogenic effects of nitric oxide.

Modification of protein moiety of human low density lipoprotein by hypochlorite generates strong platelet agonist.

Conflicting reports exist about the effects of mildly or extensively oxidized low density lipoproteins (LDLs) on the reactivity of human platelets. This platelet response is mainly caused by modification of the protein and lipid moiety, giving rise to very differently modified species with hardly predictable properties. The aim of this study was to prepare oxidized LDL with modifications essentially restricted to the protein moiety and to determine the eventual platelet responses. We treated LDL at 0 degrees C for 10 minutes with a 60- to 1000-fold molar excess of sodium hypochlorite in borate buffer in the presence of the radical scavenger butylated hydroxytoluene. Under these conditions, neither fragmentation of apolipoprotein B-100 nor formation of LDL aggregates was observed, and lipid oxidation products did not exceed the amount present in untreated LDLs. The degree of modification and the respective effects on platelet function were highly reproducible. Hypochlorite-modified LDLs act as strong platelet agonists, inducing morphological changes, dense granule release, and irreversible platelet aggregation. The evoked platelet effects are completely suppressed by inhibitors of the phosphoinositide cycle but not by EDTA or acetylsalicylic acid. Most likely, these effects are transmitted via high-affinity binding to a single class of sites, which does not recognize native or acetylated LDL. Obviously, modified lysines, and the secondary lipid modifications derived from them, are not essential for this interaction. We conclude that bioactive oxidized lipids are not directly involved in the stimulation of platelets by hypochlorite-modified LDLs.

Stimulating effect of biologically modified low density lipoproteins on ADP-induced aggregation of washed platelets persists in absence of specific binding.

Oxidized low density lipoproteins are closely associated with atherosclerosis and also might be directly involved in thrombosis because they have been shown to mediate a stimulating effect on human platelets. In this work, we used biologically modified low density lipoproteins (i.e., low density lipoproteins sufficiently oxidized to show specificity for the macrophage scavenger receptor system) to examine if specific binding of the oxidized apolipoprotein moiety to the platelet surface is a prerequisite for the platelet-stimulating effects reported by other authors. We find that biologically modified low density lipoproteins show specific binding to human platelets (K(d)=5.83+/-0.4 microg/mL, 3850+/-620 sites/platelet) and strongly augment both ADP- and thrombin-induced aggregation of washed platelets. Maleylated albumin, an antagonist of oxidized low density lipoproteins binding to all currently classified scavenger receptors, is able to reduce platelet oxidized low density lipoproteins binding to background levels. Nevertheless, maleylated albumin is not able to exert any kind of normalizing effect on the augmented ADP-induced aggregation response observed in the presence of biologically modified low density lipoproteins. From these data, we conclude that specific binding of oxidatively modified apolipoprotein B to the platelet surface is not essential to the process of platelet stimulation. Therefore, we conclude that these stimulating effects may be mediated by unidentified compounds formed in the lipid phase of the lipoproteins.

Oxidized low-density lipoprotein is localized in the ventricles of hearts from patients with coronary heart disease.

The present study was designed to investigate whether oxidized low-density lipoprotein is accumulated in the left and right ventricular walls of patients with coronary heart disease (n=10) compared with patients with dilated cardiomyopathy (n=9) or healthy heart donors (controls, n=5). Sections from both ventricles of explanted hearts and coronary arteries of the same patients were analyzed by semiquantitative immunohistochemistry for the presence of oxidized low-density lipoprotein. Oxidized low-density lipoprotein was enriched in the left and right ventricular walls from coronary heart disease patients compared with patients with dilated cardiomyopathy (P=0.0012 for left ventricle and P=0.103 for right ventricle) or controls (P=0.0012 for the left ventricle and P<0.05 for the right ventricle). The accumulation of oxidized low-density lipoprotein was higher in the left than in the right ventricles in all three groups. Positive immunoreactivity for oxidized low-density lipoprotein was mainly identified in the endocardium and the subendocardial areas of the ventricles and co-localized with macrophages. Accumulation of oxidized low-density lipoprotein in the ventricles significantly correlated with the enrichment in the respective coronary arteries, whereas only poor correlations were observed between various hemodynamic parameters and ventricular oxidized low-density lipoprotein accumulation. Ventricular accumulation of oxidized low-density lipoprotein seems to be a generalized pathophysiological process which does not exclusively involve the coronary arteries. Higher oxidative stress in combination with impaired oxygen supply in the endocardium could have favored low-density lipoprotein deposition and oxidation.

Urea induces macrophage proliferation by inhibition of inducible nitric oxide synthesis.

BACKGROUND: Atherosclerosis is a major cause of morbidity and mortality in chronic renal failure and is associated with the proliferation of macrophages within atherosclerotic lesions. METHODS: Because the progression of atherosclerosis as a consequence of decreased nitric oxide synthesis has been described, we investigated the correlation between the inhibition of inducible nitric oxide synthase (iNOS) by urea, macrophage proliferation as assayed by cell counting, tritiated thymidine incorporation and measurement of cell protein, and macrophage apoptosis. RESULTS: Urea induces a dose-dependent inhibition of inducible nitric oxide synthesis in lipopolysaccharide-stimulated mouse macrophages (RAW 264.7) with concomitant macrophage proliferation. Macrophage proliferation, as determined by cell counting, became statistically significant at 60 mM urea, corresponding to a blood urea nitrogen level of 180 mg/100 ml, concentrations seen in uremic patients. iNOS protein expression showed a dose-dependent reduction, as revealed by immunoblotting when cells were incubated with increasing amounts of urea. The decrease of cytosolic DNA fragments in stimulated macrophages incubated with urea shows that the proliferative actions of urea are associated with a decrease of NO-induced apoptosis. CONCLUSIONS: Our data demonstrate that the inhibition of iNOS-dependent NO production caused by urea enhances macrophage proliferation as a consequence of diminished NO-mediated apoptosis.

Human platelets exclusively bind oxidized low density lipoprotein showing no specificity for acetylated low density lipoprotein.

The widely studied macrophage scavenger receptor system is known to bind both acetylated low density lipoprotein and oxidized low density lipoprotein. Although only the latter ligand has been shown to occur in vivo, acetylated low density lipoprotein is often used to evaluate the contribution of scavenger receptors to different (patho)physiologic processes, assuming that all existing subtypes of scavenger receptors recognise both lipoproteins. In the present work, we identify human platelets as the first natural cell type to bind oxidized low density lipoprotein without showing specificity for acetylated low density lipoprotein. Consequently, platelets possess exclusive receptor(s) for oxidized low density lipoprotein distinct from the 'classical' scavenger receptor AI/AII. From the data presented in this work, we conclude that the class B scavenger receptor CD36 (GPIV) is responsible for this exclusive oxidized low density lipoprotein binding.

Colocalization of gold-labeled LDL and fibrinogen on platelets: enhanced fibrinogen binding induced by LDL.

Low-density lipoproteins (LDL) specifically bind to the human platelet integrin-alpha IIb and -beta 3 (Koller et al., J. Biol. Chem. 264: 12412-12418, 1989). We show by electron microscopy (EM) that gold (Au)-labeled LDL bind to sites randomly distributed on the surface of platelets in suspension. Within a few minutes, mobile ligand-receptor complexes are translocated from the surface to the open canalicular system (OCS), which finally centralizes as a broad belt. Binding and translocation of Au-LDL are independent of stimulation of platelets by ADP and are completely reversible. Au-fibrinogen shows a strikingly similar, though agonist-dependent, redistribution behavior. Platelets are markedly activated by LDL. This activation is initiated by the binding of LDL to the plasma membrane receptor, and receptor internalization is probably not required for the activation but may instead be one of its consequences. Coincubation with Au-LDL and Au-fibrinogen results in more pronounced activation. The amount of OCS-localized ligands is significantly increased, most likely reflecting enhanced receptor recycling. The two ligands show a tendency to segregate in separate clusters, indicating differences in their postbinding pathways.

Spectrophotometric determination of ascorbic acid and dehydroascorbic acid.

We present a method for measuring ascorbic acid in methanol/trichloroacetic acid extracts prepared from human plasma after enzymatic oxidation of ascorbic acid to dehydroascorbic acid by ascorbate oxidase. Samples were assayed by spectrophotometrically monitoring the kinetics of the concentration-dependent absorbance changes of dehydroascorbic acid with phosphate-citrate-methanol buffers. Ascorbic acid was determined as the difference between dehydroascorbic acid and total ascorbic acid content. The detection limit was < 0.5 mumol/L. The calibration curve was linear (r > 0.995) over the range 0-1000 mumol/L. Analytical recovery of ascorbic acid added to plasma was 93-105%. The between-day variance was < 7%. Comparison of the spectrophotometric determination (y) with a chromatographic procedure (x) gave y = 1.02x - 0.653 (Sylx = 3.61) over the range of physiologically relevant concentrations. Total analysis time is < 10 min per sample and allows the simultaneous analysis of multiple samples.