José J. Zamorano-León

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.

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.

Modifications by Olmesartan medoxomil treatment of the platelet protein profile of moderate hypertensive patients.

Olmesartan medoxomil is a new angiotensin II receptor blockers (ARB) which exhibits pleiotropic effects that are not fully understood. Our aims were: i) to determine the effect of Olmesartan medoxomil on blood pressure, lipid profile and renal functionality in moderately hypertensive patients with non‐controlled blood pressure, ii) to determine if Olmesartan medoxomil may exert anti‐inflammatory effects and modify the expression profile of platelet proteins. Thirteen moderate hypertensive patients with non‐controlled systolic blood pressure (SBP) and renal function classified as Kidney Disease Outcome Quality Initiative stage 2–3 were included. Patients were treated with Olmesartan medoxomil (20 mg/day) for 6 months. SBP, proteinuria and the plasma levels of cholesterol and low density lipoprotein (LDL)‐cholesterol were reduced after the treatment. Olmesartan medoxomil did not modify the circulating plasma levels of a number of proteins associated with inflammation, but reduced the expression level of different platelet proteins including tropomyosin‐β chain isotypes 3 and 4, serotransferrin isotypes 1 to 5, the leukocyte elastase inhibitor and the chloride intracellular channel‐protein isotype 1. The expression of the gelsolin precursor isotype 4 was increased in the platelets after the treatment. In summary, Olmesartan medoxomil reduced SBP, total and LDL‐cholesterol plasma levels and urinary protein excretion and induced changes in the expression of platelet proteins which may be related to some action of the drug at the megakaryocyte level.

Impact of Clopidogrel and Aspirin Treatment on the Expression of Proteins in Platelets from Type-2 Diabetic Patients with Stable Coronary Ischemia

The purpose of this study was to compare the effect of dual antiplatelet therapy [clopidogrel + aspirin (ASA)] with respect to ASA on the protein expression of platelets from controlled type-2 diabetic patients with stable coronary ischemia. Patients had been taking ASA (100 mg day) and they were randomized to receive (n = 29) or not (n = 28) 75 mg day clopidogrel for 12 ± 2 weeks in a blind form. Protein expression was analyzed by two-dimensional electrophoresis and mass spectrometry. The protein expression of a limited number of proteins such as actin-binding protein isotypes 2 and 5, lactate dehydrogenase, serotransferrin isotype 4, protein disulfide isomerase-A3 isotype 1, fibrinogen beta chain isotype 5, Ras-related protein Rab-7b isotypes 1 and 6, and immunoglobulin heavy chain was changed after dual antiplatelet therapy. Plasma level of platelet factor 4 (PF4), an in vivo marker of platelet activity, was not different between both groups. These changes suggest lower platelet reactivity after dual antiplatelet therapy in the studied patients. However, the variation in platelet proteome was lower than it would be initially expected, taking into account the apparent clinical beneficial effects of dual antiplatelet therapy. PF4 plasma level was not further decreased in the platelets treated for a longer time than 9-12 months with ASA + clopidogrel, as compared with ASA alone.

Plasma desmoplakin I biomarker of vascular recurrence after ischemic stroke

J. Neurochem. (2012) 121, 314–325.

New circulating biomarkers for predicting cardiovascular death in healthy population.

There is interest to analyse newer biomarkers to identify healthy individuals at risk to develop cardiovascular disease (CVD) incidents and death. To determine in healthy individuals new circulating protein biomarkers, whose systemic levels may be associated with the risk of future development of CVD incidents and death. The study was performed in 82 individuals from the Malmö Diet and Cancer study cohort, free from CVD of whom 41 developed CVD and 41 did not. Plasma proteins related to inflammation and thrombo‐coagulating processes were analysed. α1‐antitrypsin isotype 3 plasma levels were significantly higher while apolipoprotein J plasma levels were lower in participants that developed CVD incidents than those that did not develop acute cardiovascular episode. Of 82 participants, 17 died by CVD causes. There were proteins whose expression in plasma was significantly higher in participants suffering CVD death as compared with those that did not die by CVD. These proteins included: fibrinogen β‐chain isotypes 1 and 3, fibrinogen‐γ‐chain isotype 2, vitamin D‐binding protein isotypes 1, 2 and 3, α1‐antitrypsin isotypes 3 and 6, haptoglobin isotypes 3,4,5 and 5, haemopexin isotypes 1 and 2, and Rho/Rac guanine nucleotide exchange factor 2. Moreover, apolipoprotein J plasma levels were found lower in participants that died by cardiovascular cause. Association between plasma levels of proteins and CVD death was independent of age, gender, conventional risk factors and plasma C‐reactive protein levels. Several protein plasma levels and protein isotypes related to inflammation and thrombo‐coagulating phenomena were independently associated with the risk of future CVD death.

Effects of Coronary Prestenting Platelet Inhibition on Coronary Poststenting Inflammation

The aim of this study was to analyze the effect of 2 antiplatelet regimens on the inhibition of GP IIb/IIIa-dependent platelet activation and their association with the poststenting inflammatory response. Seventeen patients with acute myocardial infarction were divided into 2 groups: (A) clopidogrel plus tirofiban infusion administered together during inclusion (n = 10); (B) clopidogrel administered at inclusion and followed 2 hours after by tirofiban (n = 7). Blood samples were obtained at inclusion and at 24 and 48 hours after stenting. Before stenting, a greater reduction of GP IIb/IIIa-dependent platelet activation was found in both groups, although it was greater in group A than in group B. This statistical difference was not observed at 24 and 48 hours after the procedure. At 48 hours after stenting, interleukin-6, interleukin-10, soluble intracellular adhesion molecule-1, and soluble CD40 ligand plasma values were not different between experimental groups. By proteomics, different isoforms of the following proteins were identified: alpha 1-antitrypsin (ATT-1), fibrinogen gamma chain, apolipoprotein A-IV, apolipoprotein A-I, vitamin D binding protein, haptoglobin, and serotransferrin. At 48 hours after stenting, only the plasma expression of the ATT-1 isoform 5 was significantly increased in group A compared with group B. In conclusion, a greater inhibition of GP IIb/IIIa-dependent platelet activation before stenting was not correlated with a different inflammatory activity early after stenting.

Soluble guanylate cyclase β1-subunit expression is increased in mononuclear cells from patients with erectile dysfunction

The aim was to determine in circulating mononuclear cells from patients with erectile dysfunction (ED), the level of expression of endothelial nitric oxide synthase (eNOS), soluble guanylate cyclase (sGC) β1-subunit and phosphodiesterase type-V (PDE-V). Peripheral mononuclear cells from nine patients with ED of vascular origin and nine patients with ED of neurological origin were obtained. Fourteen age-matched volunteers with normal erectile function were used as control. Reduction in eNOS protein was observed in the mononuclear cells from patients with ED of vascular origin but not in those from neurological origin. Although sGC β1-subunit expression was increased in mononuclear cells from patients with ED, the sGC activity was reduced. However, only the patients with ED of vascular origin showed an increased expression of PDE-V. This work shows for the first time that, independently of the aetiology of ED, the expression of sGC β1-subunit was increased in circulating mononuclear cells; however, the expression of both eNOS and PDE-V was only modified in the circulating mononuclear cells from patients with ED of vascular origin.

Effects of Platelets on the Protein Expression in Aortic Segments: A Proteomic Approach

It is well known the effects of the vascular wall on platelet activity but little is known about the effects of platelets on the proteins expression in the vascular wall. We analyzed whether platelets may modify the protein expression in the vascular wall. We used an in vitro model coincubating human platelet rich plasma (PRP) with control and 10 ng/ml tumor necrosis factor‐α (TNF‐α)‐preincubated bovine aortic segments. 2DE, mass spectrometry and Western blot analysis were used to determine changes in the expression of proteins associated with the cytoskeleton and energetic metabolism in the aortic segments. In control healthy vascular wall, only the cytoskeleton‐related proteins expression was modified by PRP. However, when PRP was coincubated with TNF‐α pre‐stimulated aortic segments lesser number of cytoskeleton‐related proteins were modified. With respect to energetic metabolism, in control segments, PRP failed to modify any of the analyzed energetic‐related proteins. However, in TNF‐α‐preincubated segments the presence of PRP upexpressed glyceraldehyde‐3‐phosphate dehydrogenase. Moreover, by western blot experiments it was observed that in TNF‐α‐preincubated segments the expression of fructose 1,6‐bisphosphate aldolase was downregulated by platelets. However, no differences were found in the expression of triosephosphate isomerase and ATP synthase α‐chain. In addition, the activity of fructose 1,6‐bisphosphate aldolase and piruvate content was significantly reduced without modification on triosephosphate isomerase activity. In conclusion, the crosstalk between platelets and vascular wall is bidirectional and platelets regulated in the vascular wall the expression of proteins associated with the cytoskeleton and energetic metabolism, particularly in the healthy vascular wall. J. Cell. Biochem. 111: 889–898, 2010.