Ricky Y.K. Man

Oxidative modification enhances lipoprotein(a)-induced overproduction of plasminogen activator inhibitor-1 in cultured vascular endothelial cells

Elevated levels of plasma lipoprotein (a) [Lp(a)] have been considered as a strong risk factor for premature cardiovascular diseases. Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of plasminogen activators (PA). Increases in PAI-1 levels with or without a reduction in PA levels have been frequently found in coronary artery disease patients. The present paper examined the effects of oxidized Lp(a) on the production of PAI-1 in cultured human umbilical vein endothelial cells (HUVEC). Lp(a) and Lp(a)-free, low density lipoprotein (LDL) were prepared using lysine-Sepharose 4B affinity chromatography. Incubations with 10(-8) M levels of native Lp(a) moderately increased the levels of biologically active PAI-1 in post-culture medium of HUVEC compared to that with equimolar concentrations of native Lp(a)-free LDL. The release of PAI-1 induced by Lp(a) was enhanced by oxidative modification with copper ion. The stimulation of oxidized Lp(a) on PAI-1 production reached plateau in EC treated with 10-20 nM oxidized Lp(a) modified by microM CuSO4. Treatment with 0.2 micrograms/ml of actinomycin D significantly reduced native and oxidized Lp(a)-induced PAI-1 overproduction in EC. Increases in the steady state levels of PAI-1 mRNA were detected in native or oxidized Lp(a)-treated EC. The effect of Lp(a)-free oxidized LDL on PAI-1 production was significantly weaker than the equimolar amount of oxidized Lp(a) but stronger than that of native LDL. Treatments with oxidized Lp(a) increased cell-associated PAI-1 to a similar extent as that in native Lp(a)-treated EC. The results of the present paper demonstrate that oxidative modification enhances Lp(a)-induced PAI-1 production in vascular endothelial cells at RNA transcription level, which suggests that oxidization potentially amplifies the anti-fibrinolytic and thrombotic effect of Lp(a).

Lysophosphatidylcholine accumulation in the ischemic canine heart

The production of cardiac arrhythmias and the elevation of lysophosphatidylcholine level in the ischemic myocardium have been well-documented in a number of studies. However, the relationship between the production arrhythmias and the elevation of tissue lysophosphatidylcholine level was not reported. In this study, the lysophosphatidylcholine level and the occurrence of cardiac arrhythmias in the ischemic canine heart were monitored. A temporal relationship between the accumulation of lysophosphatidylcholine and the occurrence of arrhythmias was established after five hr of ischemia. A significant elevation of lysophosphatidylcholine was detected at three hr of ischemia without the occurrence of arrhythmias. The results indicate that cardiac arrhythmias did not cause the elevation of lysophosphatidylcholine and if lysophospholipids are causally related to the arrhythmias that a critical level of the lysophospholipid must accumulate in order to elicit electrophysiological alterations.

Alterations of phospholipids in ischemic canine myocardium during acute arrhythmia

Myocardial ischemia was produced in the left ventricle of the canine heart by a Harris two-stage occlusion of the left anterior descending coronary artery. The lipid content in the ischemic myocardium was analyzed and compared with the control tissue. No significant change in total phospholipid and cholesterol was detected. A 2-fold elevation in the levels of the major lysophospholipids was observed during acute ventricular arrhythmias at 24hr after the onset of ischemia. Such increases were not caused by preferential hydrolysis of phospholipid plasmalogens from the parent phospholipids.

Lysophosphatidylcholine-induced arrhythmias and its accumulation in the rat perfused heart

1 The tissue level of lysophosphatidylcholine (LPC) was determined in rat hearts perfused with a solution containing 5 μm LPC. The relationship between LPC accumulation and the severity of arrhythmias produced was examined. 2 The accumulation of LPC was dependent on the perfusion time and this accumulation was associated with the occurrence of severe arrhythmias. A positive correlation between the tissue LPC content and the arrhythmia score was found (P < 0.01). 3 No consistent alteration in total phospholipid, phosphatidylcholine or cholesterol content was found. This suggests that LPC‐induced arrhythmias are not associated with alterations of major lipid components in the heart. 4 When severe arrhythmias occurred in the presence of LPC in the rat perfused heart, less than 2% of total tissue phospholipid was in the form of LPC. 5 The positive correlation between LPC accumulation and the occurrence of arrhythmias suggests a cause and effect relationship of LPC with cardiac arrhythmias in the rat perfused heart. However, in the ischaemic heart, other biochemical factors can contribute, to different degrees, to ischaemia‐induced cardiac arrhythmias.

Biphasic modulation of platelet phospholipase A2 activity and platelet aggregation by mepacrine (quinacrine).

The modulation of rat platelet phospholipase A2 (phosphatide 2-acylhydrolase, EC 3.1.1.4) and rat platelet aggregation by mepacrine was investigated. The 2-acyl specificity of phospholipase A2 activity was confirmed by using 1-[14C]palmitoyl-2-[3H]arachidonylphosphatidylcholine as substrate. Under optimal pH, phospholipase A2 activity was not affected by aspirin but was inhibited by indomethacin. Contrary to previous reports, a biphasic modulatory role of mepacrine on phospholipase A2 activity and platelet aggregation was demonstrated. The data suggest that platelet aggregation is mediated via phospholipase A2.

Ganoderma lucidum inhibits inducible nitric oxide synthase expression in macrophages

Nitric oxide (NO) is a principal mediator in many physiological and pathological processes. Overproduction of NO via the inducible nitric oxide synthase (iNOS) has cytotoxic effect through the formation of peroxynitrite with superoxide anion. The iNOS is mainly expressed in macrophages and is able to produce large amount of NO. The expression of iNOS is mainly regulated at the transcriptional level. The iNOS-mediated NO production plays a role in the development of atherosclerosis. Ganoderma lucidum (G. lucidum, Linzhi or Reishi) is a traditional herbal medicine which is commonly used as health supplement. Several studies have demonstrated its effectiveness against cancer, immunological disorders and cardiovascular diseases. The objective of the present study was to investigate the effect of G. lucidum on iNOS-mediated NO production in macrophages. Human monocytic cell (THP-1) derived macrophages were incubated with lipopolysaccharide (LPS) for 24 h. Such treatment significantly stimulated NO production (253% versus the control). Such a stimulatory effect was resulted from increased iNOS mRNA expression (270% versus the control) and iNOS activity (169.5% versus the control) in macrophages. The superoxide anion level was also elevated (150% versus the control) in LPS-treated macrophages. Treatment of macrophages with G. lucidum extract (100 μg/ml) completely abolished LPS-induced iNOS mRNA expression and NO production. Such an inhibitory effect of G. lucidum was mediated via its antioxidant action against LPS-induced superoxide anion generation in macrophages. These results suggest that G. lucidum may exert a therapeutic effect against atherosclerosis via ameliorating iNOS-mediated NO overproduction in macrophages.

Phosphatidylcholine metabolism in ischemic and hypoxic hearts

The rates of phosphatidylcholine biosynthesis in the isolated hamster hearts under ischemic and hypoxic conditions were examined. Global ischemia was produced by perfusion of the heart with a reduced flow, whereas hypoxia was produced by perfusion with a N2-saturated buffer. A 51% reduction in the biosynthesis of phosphatidylcholine was observed in the ischemic heart. The reduction was caused by a severe decrease in ATP level which resulted in a diminished conversion of choline into phosphocholine. A 22% reduction in the biosynthetic rate of phosphatidylcholine was also detected in the hypoxic heart. The reduction was caused by a diminished level of CTP which resulted in a decreased conversion of phosphocholine to CDP-choline. No compensatory mechanism was triggered during ischemia, but the CTP: phosphocholine cytidylyltransferase activity was enhanced in the hypoxic heart. Our results demonstrate the possible rate-limiting role of choline kinase and reconfirm the regulatory role of the cytidylyltransferase in the biosynthesis of phosphatidylcholine. (Mol Cell Biochem116: 53–58, 1992)

The association of lysophosphatidylcholine with isolated cardiac myocytes

The ability of exogenous lysophosphatidylcholine to produce electrophysiological derangements and cardiac arrhythmias in the heart has been documented. The action of lysophosphatidylcholine is thought to be mediated via its association with the membrane. The present study examined the nature of the association of lysophosphatidylcholine with isolated rat myocyte membrane. The association was studied by incubating myocytes in a lysophosphatidylcholine-containing medium. The association of lysophosphatidylcholine with the myocyte sarcolemma was not affected by palmitic acid and glycerophosphocholine but was reduced by platelet-activating factor (PAF). The addition of albumin (5 mg/mL) at the end of the incubation period effectively removed the lysophosphatidylcholine from the myocytes. Our results suggest that most of the lysophosphatidylcholine in isolated myocytes was associated preferentially with the outer leaflet of the myocyte sarcolemma. This type of association might be responsible for the lysophosphatidylcholine-induced electrophysiological alterations in the heart.

Mechanisms of the coronary vascular effects of platelet-activating factor in the rat perfused heart.

1 In a previous study it was demonstrated that bolus injections of platelet‐activating factor (PAF) in the rat perfused heart resulted in coronary vasodilatation, vasoconstriction or the combination of both, depending on the amount of PAF that was injected. In the present study, the mechanisms of these coronary vascular effects of PAF in the rat perfused heart were investigated. 2 Pretreatment of the rat perfused heart with the PAF antagonists FR‐900452 or BN‐52021 did not affect the vasodilator effect of PAF but eliminated the vasoconstrictor effect of PAF. FR‐900452 had no effect on the vasoconstrictor response to leukotriene C4 (LTC4) or LTD4. 3 The cyclo‐oxygenase inhibitor, indomethacin, did not modify the coronary vascular effects of PAF. However L‐649,923 (a leukotriene antagonist) and MK‐886 (a leukotriene synthesis inhibitor) eliminated both the vasodilator and vasoconstrictor effects of PAF. 4 When leukotrienes were administered by bolus injection in the rat perfused heart, LTB4 produced vasodilatation while LTC4 and LTD4 produced vasoconstriction. L‐649,923 blocked both the vasodilator and vasoconstrictor effects of the leukotrienes tested. 5 The results suggest that lipoxygenase products are responsible for both the vasodilator and vasoconstrictor actions of PAF in the coronary vasculature of the rat perfused heart while the cyclo‐oxygenase products do not play a significant role. The ineffectiveness of PAF antagonists in blocking the vasodilatation produced by PAF is compatible with the concept that there may be multiple PAF receptors.

Effects of lysophosphoglycerides on cardiac arrhythmias

The accumulation of lysophosphoglycerides has been implicated as an important biochemical factor for cardiac arrhythmias. Recently, we demonstrated that lysophosphatidylcholine caused cardiac arrhythmias in the isolated hamster heart. In this study, the arrhythmogenic nature of various lysophosphoglycerides with respect to acyl chain lengths and base groups were assessed. We demonstrated that all naturally occurring lysolipids tested were arrhythmogenic at 0.05-0.10 mM. Arrhythmias were also observed with Triton X-100 or sodium laurylsulfate at 0.05-0.10 mM. Our data suggests that no correlation exists between the arrhythmogenic nature of the lysolipids and their critical micelle concentrations. We postulate that arrhythmias are produced by the detergent effect of lysophosphoglycerides.