Ryszard Korbut

Corticosteroids inhibit prostaglandin release from perfused mesenteric blood vessels of rabbit and from perfused lungs of sensitized guinea pig

Infusion of norephinephrine (NE) (1 - 3 mug/ml/min) into the isolated mesenteric vascular preparation of rabbit resulted in a rise in perfusion pressure, which was associated with the release of prostaglandin E-like substance (PGE) at a concentration of 2.81 +/- 0.65 ng/ml in terms of PGE2. Indomethacin (3 mug/ml) abolished the NE-induced release of PGE. Arachidonic acid (0.2 mug/ml) in the presence of indomethacin did not restore the NE-induced release of PGE. Hydrocortisone (10 - 30 mug/ml) and dexamethasone (2 - 5 mug/ml) also inhibited the NE-induced release of PGE. The inhibitory action of both corticosteroids was abolished by arachidonic acid (0.2 mug/ml). Antigen-induced release of a prostaglandin-like substance (PGs) (43.1 +/- 3.8 ng/ml in terms of PGE2 and a rabbit aorta contracting substance (RCS) from perfused lungs of sensitized guinea pigs was completely abolished by indomethacin (5 mug/ml) or by hydrocortisone (100 mug/ml). Indomethacin, however, increased histamine release up to 280% of the control level, which was 470 +/- 54 ng/ml, while hydrocortisone diminished histamine release down to 30% of the control level. A superimposed infusion of arachidonic acid (1 mug/ml) into the pulmonary artery reversed the hydrocortisone-induced blockade of the release of RCS and PGs. It may be concluded that corticosteroids neither inhibit prostaglandin synthetase nor influence prostaglandin transport through the membranes but they do impair the availability of the substrate for the enzyme.

Coronary Artery Superoxide Production and Nox Isoform Expression in Human Coronary Artery Disease

Background—Oxidative stress plays important role in the pathogenesis of atherosclerosis and coronary artery disease (CAD). We aimed to determine the sources and selected molecular mechanisms of oxidative stress in CAD. Methods and Results—We examined basal and NAD(P)H oxidase-mediated superoxide (O2˙−) production using lucigenin chemiluminescence, ferricytochrome c and dihydroethidium fluorescence in human coronary arteries from 19 CAD and 17 non-CAD patients undergoing heart transplantation. NAD(P)H oxidase subunits and xanthine oxidase expression were measured. Superoxide production was greater in coronary arteries from patients with CAD, even in vessels without overt atherosclerotic plaques, and was doubled within branching points of coronary arteries. Studies using pharmacological inhibitors and specific substrates showed that NAD(P)H oxidases (60%) and xanthine oxidase (25%) are primary sources of O2˙− in CAD. Losartan significantly inhibited superoxide production in coronary arteries. NAD(P)H oxidase activity and protein levels of the NADPH oxidase subunits p22phox, p67phox, and p47phox were significantly increased in CAD, as were mRNA levels for p22phox and nox2, and no NAD(P)H oxidase subunit mRNA levels correlated with NAD(P)H oxidase activity in vessels from individual patients. Activity and protein expression of xanthine oxidase were increased in CAD, whereas xanthine dehydrogenase levels were not changed. Conclusions—Increased expression and activity of NAD(P)H oxidase subunits and xanthine oxidase, in part mediated through angiotensin II and PKC-dependent pathways, are important mechanisms underlying increased oxidative stress in human coronary artery disease.

Effects of prolonged oral supplementation with l-arginine on blood pressure and nitric oxide synthesis in preeclampsia.

Background  Several lines of evidence point to the dysfunction of the endothelial l‐arginine–NO system in preeclampsia. We investigated the influence of dietary supplementation with l‐arginine on blood pressure and biochemical measures of NO production in women with preeclampsia in prospective, randomized, placebo‐controlled study.

Inhibition of five lipoxygenase activating protein (FLAP) by MK-886 decreases atherosclerosis in apoE/LDLR-double knockout mice

Background  Recent reports point to an important role of leukotrienes in atherogenesis. Leukotrienes are produced by 5‐lipoxygenase co‐operating with five lipoxygenase activating protein (FLAP). We hypothesized that MK‐886, an inhibitor of FLAP, could attenuate the development of atherosclerosis in the atherogenic apolipoprotein E/low density lipoprotein receptor (apoE/LDLR) double knockout (DKO) mouse model.

Mechanisms of oxidative stress in human aortic aneurysms — Association with clinical risk factors for atherosclerosis and disease severity

Aortic abdominal aneurysms (AAA) are important causes of cardiovascular morbidity and mortality. Oxidative stress may link multiple mechanisms of AAA including vascular inflammation and increased metalloproteinase activity. However, the mechanisms of vascular free radical production remain unknown. Accordingly, we aimed to determine sources and molecular regulation of vascular superoxide (O2•−) production in human AAA. Methods and results AAA segments and matched non-dilated aortic samples were obtained from 40 subjects undergoing AAA repair. MDA levels (determined by HPLC/MS) were greater in plasma of AAA subjects (n = 16) than in risk factor matched controls (n = 16). Similarly, superoxide production, measured by lucigenin chemiluminescence and dihydroethidium fluorescence, was increased in aneurysmatic segments compared to non-dilated aortic specimens. NADPH oxidases and iNOS are the primary sources of O2•− in AAA. Xanthine oxidase, mitochondrial oxidases and cyclooxygenase inhibition had minor or no effect. Protein kinase C inhibition had no effect on superoxide production in AAA. NADPH oxidase subunit mRNA levels for p22phox, nox2 and nox5 were significantly increased in AAAs while nox4 mRNA expression was lower. Superoxide production was higher in subjects with increased AAA repair risk Vanzetto score and was significantly associated with smoking, hypercholesterolemia and presence of CAD in AAA cohort. Basal superoxide production and NADPH oxidase activity were correlated to aneurysm size. Conclusions Increased expression and activity of NADPH oxidases are important mechanisms underlying oxidative stress in human aortic abdominal aneurysm. Uncoupled iNOS may link oxidative stress to inflammation in AAA. Oxidative stress is related to aneurysm size and major clinical risk factors in AAA patients.

Thrombolytic action of ticlopidine: possible mechanisms

Ticlopidine (Ticlide), an anti-platelet drug with a broad scope of clinical applications, is claimed to be an antagonist of adenosine diphosphate on platelet receptors. In vitro this antagonism cannot be demonstrated. Ex vivo it is detectable many hours after oral administration of the drug, perhaps subsequently to its biotransformation to an unknown metabolite. Here, we report for the first time that in patients with peripheral arterial disease and in cats with extracorporal circulation ticlopidine evokes instantaneous thrombolytic or fibrinolytic effects which are not associated with inhibition of platelet aggregation. Shortening of euglobulin clot lysis time and increase in plasma levels of tissue plasminogen activator were observed 1-2 h after oral ingestion of ticlopidine at a single dose of 500 mg. In cats ticlopidine produced instantaneous anti-thrombotic and thrombolytic effects at doses of 0.3-1 mg/kg and 10-15 mg/kg i.v., respectively. Thrombolysis by ticlopidine (10 mg/kg i.v.) was comparable to that by prostacyclin at a dose of 0.3 microgram/kg i.v. Ticlopidine at a concentration of 100 microM increased endothelial thromboresistance in vitro. The drug did not inhibit the activity of cyclooxygenase-1 or 12-lipoxygenase while it inhibited lipid autooxidation (IC50 = 18 microM) in rat liver microsomes. Our data point to a possibility that the therapeutic efficacy of ticlopidine might be associated not only with its delayed anti-platelet effects but also with its immediate thrombolytic action which is likely to be mediated by endothelial prostacyclin and tissue plasminogen activator rather than by platelet mechanisms.

Cationic derivatives of dextran and hydroxypropylcellulose as novel potential heparin antagonists.

Cationic derivatives of dextran (Dex) and hydroxypropylcellulose (HPC) were studied as potential alternatives of protamine sulfate (PS) used in the reversal of anticoagulant activity of heparin. The modification was performed by the attachment of cationic groups to the Dex main chain or by grafting short side chains of a polycation onto HPC. The cationic derivatives of these polysaccharides were found to bind heparin with the efficiency increasing with growing degree of cationic modification. The degree of cationic modification and consequently the ζ potential of the polymers do not have to be high to achieve effective heparin binding. The size of the complexes of cationic Dex with unfractionated heparin (UFH) is a few micrometers. For complexes of cationic HPC and UFH the size is much below 1 μm, both below and above the lower critical solution temperature of HPC. None of the cationic polysaccharides studied caused hemolysis. The concentrations of the polymers inducing the aggregation of human erythrocytes in vitro were determined.

Local inflammation is associated with aortic thrombus formation in abdominal aortic aneurysms. Relationship to clinical risk factors.

Intraluminal thrombus formation in aortic abdominal aneurysms (AAA) is associated with adverse clinical prognosis. Interplay between coagulation and inflammation, characterised by leukocyte infiltration and cytokine production, has been implicated in AAA thrombus formation. We studied leukocyte (CD45+) content by flow cytometry in AAA thrombi from 27 patients undergoing surgical repair. Luminal parts of thrombi were leukocyte-rich, while abluminal segments showed low leukocyte content. CD66b+ granulocytes were the most prevalent, but their content was similar to blood. Monocytes (CD14+) and T cells (CD3+) were also abundant, while content of B lymphocytes (CD19+) and NK cells (CD56+CD16+) were low. Thrombi showed comparable content of CD14highCD16- monocytes and lower CD14highCD16+ and CD14dimCD16+, than blood. Monocytes were activated with high CD11b, CD11c and HLA-DR expression. Total T cell content was decreased in AAA thrombus compared to peripheral blood but CD8 and CD3+CD4-CD8- (double negative T cell) contents were increased in thrombi. CD4+ cells were lower but highly activated (high CD69, CD25 and HLA-DR). No differences in T regulatory (CD4+CD25+FoxP3+) cell or pro-atherogenic CD4+CD28null lymphocyte content were observed between thrombi and blood. Thrombus T cells expressed high levels of CCR5 receptor for chemokine RANTES, commonly released from activated platelets. Leukocyte or T cell content in thrombi was not correlated with aneurysm size. However, CD3+ content was significantly associated with smoking in multivariate analysis taking into account major risk factors for atherosclerosis. In conclusion, intraluminal AAA thrombi are highly inflamed, predominantly with granulocytes, CD14highCD16- monocytes and activated T lymphocytes. Smoking is associated with T cell infiltration in AAA intraluminal thrombi.

Mitochondrial Aldehyde Dehydrogenase Activation by Alda‐1 Inhibits Atherosclerosis and Attenuates Hepatic Steatosis in Apolipoprotein E‐Knockout Mice

Background Mitochondrial dysfunction has been shown to play an important role in the development of atherosclerosis and nonalcoholic fatty liver disease (NAFLD). Mitochondrial aldehyde dehydrogenase (ALDH2), an enzyme responsible for the detoxification of reactive aldehydes, is considered to exert protective function in mitochondria. We investigated the influence of Alda‐1, an activator of ALDH2, on atherogenesis and on the liver steatosis in apolipoprotein E knockout (apoE−/−) mice. Methods and Results Alda‐1 caused decrease of atherosclerotic lesions approximately 25% as estimated by “en face” and “cross‐section” methods without influence on plasma lipid profile, atherosclerosis‐related markers of inflammation, and macrophage and smooth muscle content in the plaques. Plaque nitrotyrosine was not changed upon Alda‐1 treatment, and there were no changes in aortic mRNA levels of factors involved in antioxidative defense, regulation of apoptosis, mitogenesis, and autophagy. Hematoxylin/eosin staining showed decrease of steatotic changes in liver of Alda‐1‐treated apoE−/− mice. Alda‐1 attenuated formation of 4‐hydroxy‐2‐nonenal (4‐HNE) protein adducts and decreased triglyceride content in liver tissue. Two‐dimensional electrophoresis coupled with mass spectrometry identified 20 differentially expressed mitochondrial proteins upon Alda‐1 treatment in liver of apoE−/− mice, mostly proteins related to metabolism and oxidative stress. The most up‐regulated were the proteins that participated in beta oxidation of fatty acids. Conclusions Collectively, Alda‐1 inhibited atherosclerosis and attenuated NAFLD in apoE−/− mice. The pattern of changes suggests a beneficial effect of Alda‐1 in NAFLD; however, the exact liver functional consequences of the revealed alterations as well as the mechanism(s) of antiatherosclerotic Alda‐1 action require further investigation.

Antiatherosclerotic effect of Ang- (1-7) non-peptide mimetic (AVE 0991) is mediated by inhibition of perivascular and plaque inflammation in early atherosclerosis.

Inflammation plays a key role in atherosclerosis. The protective role of angiotensin 1–7 (Ang‐(1–7)) in vascular pathologies suggested the therapeutic use of low MW, non‐peptide Ang‐(1–7) mimetics, such as AVE0991. The mechanisms underlying the vaso‐protective effects of AVE0991, a Mas receptor agonist, remain to be explored.