Petra J. Mateos-Cáceres

Effect of clopidogrel on the expression of inflammatory markers in rabbit ischemic coronary artery

Inflammation and platelet activation are critical phenomena in the setting of acute coronary syndromes. Platelets may contribute to increase ischemic injury by enhancing the inflammatory response of leukocytes and endothelial myocardial cells. Pharmacological inhibition of platelet activation prevents ischemic complications in patients with coronary diseases. Agents directed against the integrin glycoprotein IIb/IIIa (GP IIb/IIIa) receptor not only inhibit platelet aggregation but also have been demonstrated to limit the inflammatory response in acute coronary syndromes. The question then raised is if the inhibition of platelet activation by other mechanisms than the blockade of GP IIb/IIIa may also exert anti‐inflammatory effects. The aim of the present study was to analyze if clopidogrel may exert anti‐inflammatory effects during the acute phase of myocardial infarction. A ligature was placed around the left anterior descending coronary artery of New Zealand White rabbits. After 15 min of ischemia, the myocardium was reperfused and the ischemic coronary artery was isolated 24 h after the ischemia. A group of ischemic rabbits was given a single oral dose of clopidogrel (20 mg kg−1) just after the arterial occlusion and the animal was recovered. Sham‐operated animals served as control. P‐selectin expression was significantly increased in infarcted rabbits with respect to control rabbits. Clopidogrel administration reduced P‐selectin expression with respect to untreated infarcted rabbits. CD40 ligand and tissue factor expression was increased in the ischemic coronary artery and reduced after clopidogrel administration. Clopidogrel also protected endothelial nitric oxide synthase protein expression in the ischemic coronary artery, a protein that has been found downregulated under inflammatory conditions. In conclusion, inhibition of platelet activation by clopidogrel exerted anti‐inflammatory effects on the ischemic coronary artery.

Different expression of proteins in platelets from aspirin-resistant and aspirin-sensitive patients

The aim of the present study was to analyse differences in the protein expression profile between platelets from aspirin (ASA)-resistant patients and ASA-sensitive patients. We analysed platelets from 51 clinically stable coronary ischaemic patients taking ASA (100 mg/day) divided into ASA-resistant (n=25) and ASA-sensitive (n=26) based on a platelet functionality test (PFA-100). Proteins associated with cytoskeleton, energetic metabolism, oxidative stress, inflammation and cell survival were analysed by two-dimensional electrophoresis and mass spectrometry. The expression of two gelsolin precursor isotypes and one F-acting capping protein isotype was decreased in ASA-resistant platelets (p<0.05). The expression of glyceraldehyde 3-phosphate dehydrogenase was increased in the ASA-resistant platelets (1751.1 + or - 220.6 vs. 4273.3 + or - 971.7, 95% confidence interval [CI] 1815.11 to 4061.2, p=0.001). It was accompanied by a reduced expression and activity of 1,6-bisphosphate aldolase in platelets without changes in the content of pyruvate. A reduced expression of gluthathione-S-transferase and the protein disulfide isomerase isotype 1 was found in ASA-resistant platelets. The protein expression of the chloride intracellular channel isotype 1 was increased in ASA-resistant platelets (21.3 + or - 3.8 vs. 48.8 + or - 6.0, CI 29.5 to 45.95, p=0.03) while the expression of two HSP60 and two HSP71 isotypes was decreased. No changes were observed in proteins associated with inflammation. In conclusion, ASA-resistant and ASA-sensitive platelets are different in terms of the level of expression of proteins associated with mechanisms such as energetic metabolism, cytoskeleton, oxidative stress and cell survival which may be associated with their different ability to respond to ASA.

Proteomic changes related to "bewildered" circulating platelets in the acute coronary syndrome.

Acute coronary syndromes (ACS) are associated with platelet activation. The aim of the present study was to study the protein expression level associated with glycolysis, oxidative stress, cytoskeleton and cell survival in platelets obtained during an ACS. Platelets from 42 coronary ischemic patients, divided into patients admitted within 24 h after the onset of chest pain (ACS group; n=16) and patients with stable coronary ischemic disease (CAD, n=26), were analyzed using proteomics. The expression levels of proteins involved in cellular cytoskeleton (F‐actin capping, β‐tubulin, α‐tubulin isotypes 1 and 2, vinculin, vimentin and two Ras‐related protein Rab‐7b isotypes), glycolysis pathway (glyceraldehyde‐3‐phosphate dehydrogenase, lactate dehydrogenase and two pyruvate kinase isotypes) and cellular‐related antioxidant system (manganese superoxide dismutase) and even the expression and activity of glutathione‐S‐transferase were significantly reduced in platelets from ACS patients compared to CAD patients. Moreover, reduction in the expression of proteins associated with cell survival such as proteasome subunit β type 1 was also observed in ACS platelets compared with CAD platelets. Principal component and logistic regression analysis suggested the existence of factors (proteins) expressed in the platelets inversely associated with acute coronary ischemia. In summary, these results suggest the existence of circulating antioxidant, cytoskeleton and glycolytic‐“bewildered” platelets during the acute phase of a coronary event.

KCNH2 Gene Mutation: A Potential Link Between Epilepsy and Long QT-2 Syndrome

Long QT syndrome (LQTS) is closely associated with syncope, seizure, and sudden death but LQTS is frequently misdiagnosed as epilepsy. LQTS and epilepsy both belong to the group of ion channelopathies that manifest in the heart and brain. Therefore, genetic analysis of genes associated with potassium and sodium homeostasis and electrical disorders may reveal a link between epilepsy and lethal cardiac arrhythmia. Here, the authors report a young woman who suffered recurrent seizure episodes and syncopes that occurred while walking and also during rest. She showed electroencephalogram abnormalities and a pathological prolonged QTc interval in electrocardiogram. The patient and the patients asymptomatic family members underwent genetic screening of the three genes most frequently associated with LQTS: KCNQ1, KCNH2, and SCN5A. The patient and the family members did not show DNA alterations in the genes KCNQ1 and SCN5A associated with LQT-1 and LQT-3, respectively. However, the patient showed a de novo mutation 2587T→C in exon 10 of KCNH2 gene associated with LQT-2. The mutation caused a stop codon substitution (R863X) in the HERG channel, leading to a 296–amino acid deletion. The patients asymptomatic relatives did not show the KCNH2 gene mutation. R863X alteration in HERG channel may be involved in both prolonged QTc interval and epilepsy. This fact raises the possibility that R863X alteration in KCNH2-encoded potassium channel may confer susceptibility for epilepsy and cardiac LQT-2 arrhythmia.

Comparative Expression of Proteins in Left and Right Atrial Appendages From Patients With Mitral Valve Disease at Sinus Rhythm and Atrial Fibrillation

Proteomics and Atrial Appendages. Introduction: The objective was to compare by proteomics the expression of proteins associated with the cytoskeleton, energetic metabolism, and cardiac cytoprotection between left atrial appendages (LAA) and right atrial appendages (RAA) obtained from patients with mitral valve disease both in sinus rhythm (SR, n = 6) and in permanent atrial fibrillation (AF, n = 11).

New and Old Mechanisms Associated with Hypertension in the Elderly

Hypertension is a widely prevalent and important risk factor for cardiovascular diseases that increase with aging. The hallmark of hypertension in the elderly is increased vascular dysfunction. However, the molecular mechanisms by which increased blood pressure leads to vascular injury and impaired endothelial function are not well defined. In the present paper, we will analyze several mechanisms described in the scientific literature involved in hypertension in the elderly as endothelial dysfunction, increased oxygen delivery to tissues, inflammation, cellular apoptosis, and increased concentration of active metabolites. Also, we will focus on new molecular mechanisms involved in hypertension such as telomeres shortening, progenitor cells, circulating microparticles, and epigenetic factors that have appeared as possible causes of hypertension in the elderly. These molecular mechanisms may elucidate different origin for hypertension in the elderly and provide us with new targets for hypertension treatment.

Effect of C-reactive protein on Fcγ receptor II in cultured bovine endothelial cells

The major CRP (C-reactive protein) receptor on leucocytes has been identified as the low-affinity IgG receptor Fcgamma receptor II (CD32). Our aim was to assess whether inflammation may modify the presence of the CD32 receptor in BAEC (bovine aortic endothelial cells). Confocal microscopy experiments showed a weak expression of the CD32 receptor in control BAEC that was slightly increased by 10 microg/ml CRP. Incubation of BAEC with TNF-alpha (tumour necrosis factor-alpha) did not modify the fluorescence signal of CD32. Addition of CRP to TNF-alpha-incubated BAEC enhanced the fluorescence signal of the CD32 receptors. The CD32 receptors showed a perinuclear cytoplasmic localization in BAEC. An alteration of the NO (nitric oxide)-dependent vasorelaxation has been defined as endothelial dysfunction. Endothelial dysfunction has been associated with the presence of superoxide anion and with a reduction in the expression of the eNOS (endothelial NO synthase). A concentration of CRP similar to that detected in patients with cardiovascular risk (10 microg/ml) failed to modify the generation of superoxide anion stimulated by TNF-alpha. Western blot experiments showed that TNF-alpha decreased the expression of the eNOS protein, which was partially protected by treatment with 10 microg/ml CRP. The protective effect of 10 microg/ml CRP on eNOS expression in TNF-alpha-incubated BAEC was prevented by an antibody against CD32 receptors. In conclusion, the present results suggest that, although CRP has been associated with inflammation, CRP may protect the expression of eNOS protein against pro-inflammatory mediators such as TNF-alpha.

Increased cyclic guanosine monophosphate production and endothelial nitric oxide synthase level in mononuclear cells from sildenafil citrate-treated patients with erectile dysfunction.

Mononuclear cells express enzymes involved in the NO/cyclic guanosine monophosphate (cGMP) generating system, as well as PDE5. The objective of the study was to determine the effect of sildenafil citrate administration on the level of proteins involved in the NO/cGMP generating system in mononuclear cells from patients with ED. Twenty-one patients with ED (International Index of Erectile Function-Erectile Function Domain (IIEF-EFD) 17.9±0.8) were enrolled and 100 mg sildenafil citrate on-demand was administered during 12 weeks. All patients showed cardiovascular risk factors. After sildenafil citrate administration, IIEF-EFD score was improved (26±1.2 P<0.05). In the mononuclear cells, the protein level of endothelial NO synthase (eNOS) was higher after sildenafil citrate treatment. It was accompanied by reduction in the circulating plasma levels of both high-sensitive C-reactive protein and soluble intercellular adhesive molecule-1. The protein level of soluble guanylate cyclase and PDE5 did not change in the mononuclear cells after sildenafil citrate treatment. However, in the mononuclear cells exogenous NO induced a higher cGMP production after 12-weeks sildenafil citrate administration. In conclusion, in mononuclear cells from patients with ED sildenafil citrate administration increased the level of eNOS protein and increased cGMP production in response to NO. Moreover, sildenafil citrate administration reduced the plasma circulating levels of two biomarkers associated with inflammation.

Endothelin-1 induced proinflammatory markers in the myocardium and leukocytes of guinea-pigs: role of glycoprotein IIB/IIIA receptors.

AIM To assess whether endothelin-1 (ET-1) induces the in vivo expression of inflammatory-related proteins, namely cyclooxygenase-2 (COX-2) and tissue factor, in the myocardium and circulating leukocytes of guinea-pigs. The involvement of platelets was also analyzed. METHODS ET-1 (0.013 microg/min) was infused to male guinea-pigs for 45 min in the presence and absence of tirofiban, a nonpeptidic blocker of the glycoprotein IIb/IIIa receptor (GPIIb/IIIa). Tissue factor and COX-2 expression were determined by Western blot. RESULTS No changes in mean arterial pressure and heart rate were detected. ET-1-infused guinea-pigs showed a marked increase in the number of platelets expressing activated GPIIb/IIIa receptors (0.8+/-0.03% vs. 6.5+/-0.2%; P<0.05). Tirofiban (10 microg/Kg bw/min) blunted ex vivo platelet aggregation in response to ADP, although only partially reduced COX-2 and tissue factor expression in both the myocardium and leukocytes of ET-1-infused guinea-pigs. The myocardium of platelet-depleted guinea-pigs also showed a reduced COX-2 expression after ET-1 infusion (57+/-3% reduction; P<0.05). In vitro studies demonstrated that platelets (10(7) and 10(9) platelets/well) enhanced ET-1 (10(-7) mol/l)-induced COX-2 expression in heart slices. CONCLUSION ET-1 stimulated in vivo the expression of the pro-inflammatory proteins COX-2 and tissue factor in the myocardium and in leukocytes by a mechanism GPIIb/IIIa platelet receptors.

Old and new molecular mechanisms associated with platelet resistance to antithrombotics.

ABSTRACTCurrent available data show that about 5 to 40% of coronary patients treated with conventional doses of antithrombotic drugs do not display adequate antiplatelet response. Nowadays, aspirin remains the main antiplatelet therapy. However, a significant number of patients show platelet resistance to aspirin therapy, and recurrent thrombotic events occur. Combined antithrombotic therapies with thienopyridines, such as clopidogrel have been used to resolve this problem. However, clopidogrel treatment has been also associated with wide response variability, and non-responsiveness to clopidogrel also occurs in some patients. Therefore, the main question arising about the antithrombotic therapy is why particular patients do not benefit from the therapy and how they might be identified to improve their treatment. Different hypotheses have been suggested, including genetic factors, platelet heterogeneity, non-compliance and others. However, it is probably that many molecular mechanisms involved in platelet resistance to antithrombotic therapies still remains unknown. New technologies, such as proteomics and genetic, are beginning to show new unknown biological biomarkers and molecular mechanisms which may be associated with platelet antithrombotic drug resistance.