Effects of phosphodiesterase inhibitors on the inflammatory response of endothelial cells stimulated by myeloperoxidase-modified low-density lipoprotein or tumor necrosis factor alpha.

BACKGROUND: Sildenafil, vardenafil, and tadalafil are phosphodiesterase type 5 inhibitors (PDE5-Is) usually used in the treatment of erectile dysfunction (ED). Previously, we have shown the presence of myeloperoxidase-modified low-density lipoprotein (Mox-LDL) in the penises of patients with ED, and we have shown the impact of Mox-LDL on cyclic monophosphate (cGMP) level. In vitro, Mox-LDL triggered the inflammatory response by increasing the release of both interleukin 8 (IL-8) and tumor necrosis factor alpha (TNF-alpha) by endothelial cells (ECs) and monocytes respectively. OBJECTIVE: To determine whether or not the three therapeutically PDE5-Is protect against the proinflammatory effects of Mox-LDL or TNF-alpha on ECs. DESIGN, SETTING, AND PARTICIPANTS: ECs (EA.hy926) were incubated in the presence of either TNF-alpha (100 pg/ml) or Mox-LDL (200 microg/ml) with each of the three PDE5-Is (1 microM, 5 microM, and 10 microM) respectively. IL-8 production was measured in the supernatant after 48 h of incubation. MEASUREMENTS: All experiments were repeated at least three times. Statistical analysis was performed with an ANOVA. RESULTS AND LIMITATIONS: Two-way ANOVA analysis showed that TNF-alpha alone (p<0.001) or Mox-LDL alone (p<0.001) increased IL-8 production. Sildenafil, vardenafil, or tadalafil alone did not generate an increase of IL-8 production. Tadalafil in combination with Mox-LDL and TNF-alpha showed a decrease of IL-8 (p<0.05) compared with sildenafil and vardenafil. CONCLUSIONS: Among the three available PDE5-Is, tadalafil showed an additional potentially anti-inflammatory effect on relaxation. Those data could be considered for the chronic use of PDE5-Is, but extrapolations of experimental evidence to the clinical setting should be made cautiously.

Coronary stenting is associated with an acute increase in plasma myeloperoxidase in stable angina patients but not in patients with acute myocardial infarction.

BACKGROUND: Myeloperoxidase (MPO) has emerged as a critical mediator in the physiopathology of atherosclerosis from plaque formation and growth until destabilization and rupture leading to acute coronary syndrome (ACS). Using coronary stenting as a model of plaque injury, we aimed to determine the evolution of systemic MPO levels following coronary stenting in stable angina patients and in patients with acute myocardial infarction (AMI). METHODS: Plasma levels of MPO, lactoferrin, interleukin (IL)-6, C-reactive protein and PMN counts were assessed in 13 patients with Non-ST-elevation myocardial infarction (NSTEMI) (Group A) and in 29 patients with stable angina pectoris (Group B), undergoing coronary stenting. Serial blood samples were taken before angioplasty (baseline) and at 1, 6 and 24 h following initial balloon inflation. RESULTS: Following angioplasty, the overall plasma MPO levels significantly increased at 1 h in group B (120.5+/-79.0 to 166+/-79.5, p=0.003) but not in group A (121+/-63.4 to 124.7+/-76.9, p=0.753). In Group B, the increase in MPO levels at 1 h were significantly higher in the presence of complex lesions compared to patients with simple lesions (p=0.023). Lactoferrin levels showed no change over time except for a significant decrease at 6 h in group B. CONCLUSIONS: In stable angina patients, coronary stenting is associated with an acute and transient increase in plasma MPO levels, but not in lactoferrin levels, with an enhanced response in the presence of complex lesions. In contrast, we observed no changes in plasma MPO and lactoferrin levels following stenting in patients with AMI. Given its pro-inflammatory properties, the potential implication of MPO release on clinical outcome in stable patients undergoing stenting needs further investigation.

Neuraminidase alters red blood cells in sepsis.

OBJECTIVE: To investigate the influence of neuraminidase, an enzyme that cleaves sialic acid from the red blood cell (RBC) membrane, on RBC shape and biochemistry in critically ill patients. DESIGN: Prospective, observational study and in vitro laboratory study. SETTING: A 31-bed medico-surgical department of intensive care and a university-affiliated cell biology laboratory. SUBJECTS: Acutely ill patients with and without sepsis and healthy volunteers. INTERVENTIONS: Blood sampling in volunteers. MEASUREMENTS AND MAIN RESULTS: Neuraminidase activity was measured using a fluorescent assay. RBC shape was assessed by the second coefficient of dissymmetry of Pearson using a flow cytometry technique at 25 degrees C. Intraerythrocytic 2,3-diphosphoglycerate and lactate contents were also measured. Neuraminidase activity was significantly higher in septic patients compared with nonseptic patients and healthy volunteers (5.42 [4.85-6.00] vs. 4.53 [4.23-5.23] and 1.26 [0.83-1.83] mU/mL; all p < 0.05). Neuraminidase treatment modified the RBC shape in vitro in a dose-response fashion, and most of these alterations were present after 10 hours of incubation. Incubation of RBCs with phosphatidylinositol phospholipase C modified RBC shape and increased sialic acid concentrations in the supernatant, suggesting a leakage of neuraminidase from the RBC membrane. Alterations in shape were associated with increased 2,3-diphosphoglycerate (0.46 +/- 0.25 vs. 0.19 +/- 0.05 mumol/mL; p = 0.006) and lactate content (0.81 +/- 0.07 vs. 0.66 +/- 0.05 mmoL/L; p = 0.002). CONCLUSIONS: In sepsis, desialylation under the influence of increased neuraminidase activity may contribute to the alterations in RBC rheology. Inhibition of neuraminidase may represent a new therapeutic option to ameliorate RBC rheology and perhaps oxygen delivery to the cells.

Development and validation of a screening procedure for the assessment of inhibition using a recombinant enzyme.

Myeloperoxidase (MPO, E.C. 1.1.11.7) is a heme-containing enzyme that catalyses the synthesis of hypochlorous acid (HOCl) in the presence of hydrogen peroxide (H2O2) and chlorine anions. The production of HOCl is kept under strict control of neutrophils. However, in several pathological conditions, MPO is leaked in the extracellular fluid, which involves an over-production of reactive oxygen species like HOCl and promotes the damages caused by neutrophils. As a consequence, the inhibition of MPO by various agents has been investigated and a variety of molecules have been evaluated for this activity in different models. The present study aims to describe and validate a rapid screening method based on the taurine assay and using a recombinant MPO. After validation of the stock solutions used during the experiments, the amount of MPO for the completion of the reaction was measured and fixed to an optimal value. The inhibiting concentration at 50% of flufenamic acid (taken as a reference molecule) was then assessed in both a simple tube test and a microplate test and delivered similar results (1.3+/-0.2 microM vs 1.4+/-0.2 microM, P=0.2). Finally, different molecules able to inhibit MPO were evaluated in this rapid assay system providing results comparable to literature. The high throughput screening is undoubtedly a first line assessment method which affords the selection of inhibitors and permits to reduce the number of candidates for a further elucidation of the mechanism of MPO inhibition.

Conception of myeloperoxidase inhibitors derived from flufenamic acid by computational docking and structure modification.

The development of myeloperoxidase (MPO) inhibitors has been conducted using flufenamic acid as a lead compound. Computational docking of the drug and its analogs in the MPO active site was first attempted. Several molecules were then synthesized and assessed using three procedures for the measurement of their inhibiting activity: (i) the taurine assay, (ii) the accumulation of compound II, and (iii) the LDL oxidation by ELISA. Most of the synthesized molecules had an activity in the same range as flufenamic acid but none of them were able to inhibit the MPO-dependent LDL oxidation. The experiments however gave some useful indications for a rational conception of MPO inhibitors.

Inhibition of the myeloperoxidase chlorinating activity by non-steroidal anti-inflammatory drugs: flufenamic acid and its 5-chloro-derivative directly interact with a recombinant human myeloperoxidase to inhibit the synthesis of hypochlorous acid.

The present in vitro study was designed to assess the inhibition of the myeloperoxidase (MPO)/H(2)O(2)/Cl(-) system by several non steroidal anti-inflammatory drugs (NSAIDs) of the oxicam family and of nimesulide and to compare their effect with flufenamic acid in order to investigate their influence on the chlorinating activity of MPO as a protective mechanism during chronic inflammatory syndromes. The inhibition of the system was assessed by measurement of the taurine chlorination while the accumulation of compound II was used to investigate the mechanism of inhibition. The oxidation products of NSAIDs by the MPO/H(2)O(2)/Cl(-) system were identified and flufenamic acid and derivatives were also assessed in the inhibition of LDL oxidation in two models. Flufenamic acid (IC(50) = 1.1+/-0.3 microM) is the most efficient inhibitor of the MPO/H(2)O(2)/Cl(-) system and nimesulide (IC(50) = 2.1+/-0.3 microM) is more active than the other NSAIDs of the oxicam family (IC(50) = 8-12 microM). The accumulation of compound II revealed that flufenamic acid acts as an electron donor while the other NSAIDs are antagonists of chloride anions. The identification of the oxidation products confirms that flufenamic behaves like an electron donor and is directly oxidized in the 5-hydroxy-derivative but gives also the 5-chloro-derivative which similarly inhibits the MPO/H(2)O(2)/Cl(-) system. Flufenamic acid has the best inhibiting activity towards the MPO/H(2)O(2)/Cl(-) system. However, in models that assess the LDL oxidation, flufenamic acid and its derivatives were unable to properly inhibit MPO activity as the enzyme is adsorbed on macrostructures such as LDL molecules.

Triggering of inflammatory response by myeloperoxidase-oxidized LDL.

The oxidation theory proposes that LDL oxidation is an early event in atherosclerosis and that oxidized LDL contributes to atherogenesis in triggering inflammation. In contrast to the copper-modified LDL, there are few studies using myeloperoxidase-modified LDL (Mox-LDL) as an inflammation inducer. Our aim is to test whether Mox-LDL could constitute a specific inducer of the inflammatory response. Albumin, which is the most abundant protein in plasma and which is present to an identical concentration of LDL in the intima, was used for comparison. The secretion of IL-8 by endothelial cells (Ea.hy926) and TNF-alpha by monocytes (THP-1) was measured in the cell medium after exposure of these cells to native LDL, native albumin, Mox-LDL, or Mox-albumin. We observed that Mox-LDL induced a 1.5- and 2-fold increase (ANOVA; P < 0.001) in IL-8 production at 100 microg/mL and 200 microg/mL, respectively. The incubation of THP-1 cells with Mox-LDL (100 microg/mL) increased the production of TNF-alpha 2-fold over the control. Native LDL, albumin, and Mox-albumin showed no effect in either cellular types. The myeloperoxidase-modified LDL increase in cytokine release by endothelial and monocyte cells and by firing both local and systemic inflammation could induce atherogenesis and its development.

Captopril inhibits the oxidative modification of apolipoprotein B-100 caused by myeloperoxydase in a comparative in vitro assay of angiotensin converting enzyme inhibitors.

The oxidative modification of low-density lipoproteins (LDL) is a key event in the formation of atheromatous lesions. Indeed, oxidized derivatives accumulate in the vascular wall and promote a local inflammatory process which triggers the progression of the atheromatous plaque. Myeloperoxidase (MPO) has been mentioned as a major contributor to this oxidative process. It takes part in the oxidation both of lipids by chlorination and peroxidation and of apolipoprotein B-100. Based on recent observations with several anti-inflammatory and thiol-containing drugs, the present study was designed to test the hypothesis that anti-hypertensive agents from the angiotensin converting enzyme (ACE) inhibitors group inhibit the oxidative modifications of Apo B-100 caused by MPO. Captopril, ramipril, enalapril, lisinopril and fosinopril were assessed by measuring: their inhibiting effect on the MPO/H(2)O(2)/Cl(-) system, the accumulation of compound II, which reflects the inhibition of the synthesis of HOCl and the LDL oxidation by MPO in presence of several concentrations of ACE inhibitors. Only captopril, a thiol-containing ACE inhibitor, was able to significantly decrease the oxidative modification of LDL in a dose dependent manner and this by scavenging HOCl. This efficient anti-hypertensive drug therefore appears to also protect against the atherosclerotic process by this newly documented mechanism.

Thiol-containing molecules interact with the myeloperoxidase/H2O2/chloride system to inhibit LDL oxidation.

Oxidized low-density lipoproteins (LDL) accumulate in the vascular wall and promote a local inflammatory process contributing to the progression of atheromatous plaque. The key role of myeloperoxidase (MPO) in this process has been documented and the enzyme has been involved in the oxidative modification of apolipoprotein B-100 in the intima and at the surface of endothelial cells. As the inhibition of this last phenomenon could be of relevance in pharmacological interventions, thiol-containing molecules such as glutathione, captopril, and N-acetylcysteine (NAC) and its lysinate salt (NAL) were tested in this system and their properties were compared with those of flufenamic acid (control). This last compound already demonstrated an inhibition of the production of HOCl by MPO and a more intense inhibition of MPO activity than glutathione, NAC, NAL, and captopril. However, NAC and NAL inhibited the oxidative modification of LDL more intensively than captopril and glutathione whereas flufenamic acid had no comparable inhibiting effect. This could be related to the presence of LDL close to the catalytic site of the enzyme. NAC and NAL therefore appeared as the most efficient inhibitors probably as a consequence of their relatively small size. The relevance of such effects has to be documented by in vivo studies.