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Background: Mitochondria have been involved in host defense upon viral infections. Factor Xa (FXa), a coagulating factor, may also have influence on mitochondrial functionalities. The aim was to analyze if in human pulmonary microvascular endothelial cells (HPMEC), the SARS-CoV-2 (COVID-19) spike protein subunits, S1 and S2 (S1+S2), could alter mitochondrial metabolism and what is the role of FXA., Methods: HPMEC were incubated with and without recombinants S1+S2 (10 nmol/L each)., Results: In control conditions, S1+S2 failed to modify FXa expression. However, in LPS (1  μ g/mL)-incubated HPMEC, S1+S2 significantly increased FXa production. LPS tended to reduce mitochondrial membrane potential with respect to control, but in higher and significant degree, it was reduced when S1+S2 were present. LPS did not significantly modify cytochrome c oxidase activity as compared with control. Addition of S1+S2 spike subunits to LPS-incubated HPMEC significantly increased cytochrome c oxidase activity with respect to control. Lactate dehydrogenase activity was also increased by S1+S2 with respect to control and LPS alone. Protein expression level of uncoupled protein-2 (UCP-2) was markedly expressed when S1+S2 were added together to LPS. Rivaroxaban (50 nmol/L), a specific FXa inhibitor, significantly reduced all the above-mentioned alterations induced by S1+S2 including UCP-2 expression., Conclusions: In HPMEC undergoing to preinflammatory condition, COVID-19 S1+S2 spike subunits promoted alterations in mitochondria metabolism suggesting a shift from aerobic towards anaerobic metabolism that was accompanied of high FXa production. Rivaroxaban prevented all the mitochondrial metabolic changes mediated by the present COVID-19 S1 and S2 spike subunits suggesting the involvement of endogenous FXa., Competing Interests: The authors declare that they have no conflict of interest., (Copyright © 2022 Khaoula Zekri-Nechar et al.)

Microsurgical scalp reconstruction is indicated in patients with large scalp defects. The aim of this study was to compare the outcomes of scalp reconstruction in oncologic patients reconstructed with latissimus dorsi (LD), anterolateral thigh (ALT), and omental (OM) free flaps. Thirty oncologic patients underwent scalp reconstruction with LD (10), ALT (11), and OM (9) flaps. The length of the vascular pedicle, the operation time, the possibility of a two-team approach, the length of hospital stays, the complications, and the aesthetic results were evaluated. The OM flap was the flap with the shortest vascular pedicle length with a mean of 6.26 ± 0.16 cm, compared to the LD flap, which was 12.34 ± 0.55 cm and the ALT flap with 13.20 ± 0.26 cm ( p < 0.05). The average time of surgery was 6.6 ± 0.14 h in patients reconstructed with OM, compared to the LD flap, which was 8.91 ± 0.32 h and the ALT flap with 7.53 ± 0.22 h ( p < 0.05). A two-team approach was performed in all patients for OM flaps and ALT flaps, but only in two patients reconstructed with the LD flap ( p < 0.001). In patients reconstructed with the OM flap, a very satisfactory or satisfactory result was reported in seven patients (77.8%). Eight patients reported a very unsatisfactory or unsatisfactory result with LD flap (80%) and 10 patients with ALT flap (90.9%) ( p = 0.002). The mean hospital stay after surgery was not statistically significant ( p > 0.05). As for complications, two patients reconstructed with OM flap, five LT flaps, and two ALT flaps developed complications, not statistically significant ( p = 0.235). Omental flap, latissimus dorsi flap, and anterolateral thigh flap fulfill most of the characteristics for complex scalp reconstruction. The decision on which flap to use should be based on clinical aspects of the patients taking into account that the three flaps show similar rates of complications and length of hospital stay. Regarding the aesthetic outcome, OM flap or LD flap should be considered for reconstruction of extensive scalp defects.

An inadequate platelet response to aspirin (ASA) has been identified in some patients under chronic ASA treatment. The aim of this study was to analyze if ASA-sensitive and ASA-resistant platelets have differences in their apoptotic capability. Clinically stable ischemic coronary patients who had been taking ASA (100 mg/d) for at least 9 months before inclusion were divided into ASA-resistant (n = 11) and ASA-sensitive (n = 13) groups as defined by the PFA-100 test. Platelets from ASA-sensitive patients showed higher expression of the proapoptotic proteins Bak and Bax than those from ASA-resistant patients, although only Bak protein remained different when the results were adjusted by age. In resting platelets, neither caspase-3 activity nor cytosolic cytochrome C levels were different between both experimental groups. Stimulation of platelets with calcium ionophore (10 nmol/L, A23187) increased caspase-3 activity (1.91-fold higher; P < 0.05) and cytosolic cytochrome C levels (1.84-fold higher; P < 0.05) to a higher degree in ASA-sensitive than in ASA-resistant platelets. In conclusion, ASA-sensitive platelets seem to be better prepared to undergo apoptosis during robust platelet activation.

Background: Cutaneous squamous skin cell carcinoma (SCC) is the second most frequent type of non-melanoma skin cancer and is the second leading cause of death by skin cancer in Caucasian populations. However, at present it is difficult to predict patients with poor SCC prognosis., Objective: To identify proteins with expression levels that could predict SCC infiltration in SCC arising from actinic keratosis (SCC-AK)., Methods: A total of 20 biopsies from 20 different patients were studied; 10 were SCC-AK samples and 10 were taken from normal skin. Early infiltrated SCC-AK samples were selected based on histological examination, and to determine the expression of proteins, fresh skin samples were processed by two-dimensional electrophoresis., Results: The expression levels of three proteins, namely alpha hemoglobin and heat shock proteins 27 and 70 (Hsp27 and Hsp70, respectively) were significantly increased in SCC-AK samples with respect to normal control skin. However, only the expression level of Hsp70 protein positively correlated with the level of SCC-AK dermis infiltration. Immunohistological examination suggested that increased expression of Hsp70 proteins seemed to mainly occur in the cytoplasm of keratinocytes. The increased cytoplasmic Hsp70 expression in SCC-AK was confirmed by Western blot experiments., Conclusion: Cytoplasmic expression of Hsp70 could be a potential biomarker of early infiltration of SCC arising from AK., Competing Interests: The authors declare no conflict of interest.

Objectives: To analyze the uptake of breast and cervical cancer screening according to the 2017 Spanish National Health Survey (SNHS), to compare uptake rates with those obtained in the previous SNHS 2011 and to identify predictors for the uptake for these two screening tests., Study Design: Cross-sectional study., Main Outcome Measures: Uptake rates of breast cancer and cervical cancer screening were analyzed for women aged 40-69 and aged 25-65 years, respectively. Independent variables included sociodemographic characteristics and factors related to health status and lifestyle., Results: We found that 66.8 % of women aged 40-69 years had undergone mammography in the previous two years. Positive predictors for mammography uptake were age (50-69 years); marital status (married); Spanish nationality; university education; one or more chronic diseases; no alcohol consumption; physical activity; body mass index <30 kg/m 2 ; and not smoking. We observed that 73.0 % of women aged 25-65 years had undergone cervical cytology screening in the previous three years. Positive predictors for uptake were age (25-52 years); marital status (married); Spanish nationality; middle-high educational level; no chronic diseases; no alcohol consumption; physical activity; body mass index <30 kg/m 2 ; and not smoking. There was a significant decrease in the uptake rate for breast cancer screening from the previous SNHS 2011 (OR 0.89; 95 % CI 0.83-0.94)., Conclusions: The adherence rate for mammography in Spain in 2017 was below the recommended 70 % and was significantly lower than in 2011. The figures for cervical cancer screening were over 70 % and stable over time., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Mitochondria dynamic is regulated by different proteins, maintaining a balance between fission and fusion. An imbalance towards mitochondrial fission has been associated with tumor cell proliferation. The aim of this study was to analyze whether pectin modifies the viability of human colon cancer cells and the expression of proteins involved in mitochondrial fusion and fission. The human colon carcinoma cell line HT29 cells was growth in 10% fetal bovine serum in the absence and presence of pectin. Pectin reduced HT29 cell viability in a concentration-dependent manner, reaching a plateau at 150~300 μmol/L pectin. The presence of 200 μmol/L pectin reduced the expression of dynamin-related protein-1 and increased expression of the mitochondrial fusion-associated proteins mitofusin-1 and 2. Expression of cyclin B1, a protein involved in G2/M transition, was found decreased in pectin-incubated HT29 cells. Moreover, expression of p53 protein, the amount of p53 in the nucleous and β-galactosidase activity, which are all biomarkers for cellular senescence, were significantly higher in pectin-incubated HT29 cells than in HT29 cells incubated without pectin. Expression of the protein B-cell lymphoma 2 (Bcl-2) homologous antagonist/killer was increased in response to incubation with pectin. However, incubation with pectin did not affect expression of Bcl-2-associated X protein or Bcl-2, or the caspase-3 activity. Overall, we concluded that pectin reduces the viability of human HT29 colon cancer cells, which is accompanied with a shift in the expression of proteins associated with mitochondrial dynamics towards mitochondrial fusion. Moreover, incubation with pectin favors cellular senescence over apoptosis in HT29 cells., Competing Interests: AUTHOR DISCLOSURE STATEMENT The authors declare no conflict of interest.

Context: Heat generation by brown adipose tissue (BAT) in response to temperature reduction seems to be entirely related to sympathetic nervous stimulation., Objective: To analyse if temperature reduction and norepinephrine may differently affect the expression of proteins related to energy metabolism in BAT., Materials and Methods: Isolated rats BAT was incubated with/without norepinephrine (10 -6  mol/L, 24 h at 32 °C and 37 °C)., Results: In BAT, 32 °C increased the protein expression levels of carnitine palmitoyltransferase-I and -II, mitochondrial uncoupling protein-1 (UCP-1) and the expression and activity of lactate dehydrogenase. Mitochondrial F 1 -ATP synthase α-chain expression was decreased at 32 °C compared to 37 °C. Norepinephrine and at 32 °C exposure, UCP-1 expression was increased but cytochrome-c oxidase and F 1 -ATP synthase α-chain expression was reduced with respect to 37 °C., Discussion: Sympathetic stimulation seems not to be the only factor associated with heat generation., Conclusions: Temperature reduction by itself exerts some different effects on the expression of proteins related to the energy metabolism than norepinephrine.

New strategies for tacrolimus administration that conserve its immunosuppressive effect but avoiding fluctuations in tacrolimus circulating levels are needed. The aim was to analyze if subcutaneous biodegradable tacrolimus-loaded microspheres injection promoted a significant immunosuppressive response in rats. Rats received two subcutaneous tacrolimus-loaded microspheres injections at different days, the first injection was done at day 0 and the second injection was done 12 days after. Plasma circulating levels of tacrolimus, interleukin-2 (IL-2) and calcineurin phosphatase (PP2B) activity in mononuclear cells were measured. Tacrolimus plasma levels were significantly increased from the day after tacrolimus-loaded microspheres injection and remained increased during 10days. Compared to control, plasma IL-2 levels and PP2B activity in mononuclear cells were significantly decreased during ten days. At day 12, a new subcutaneous injection of tacrolimus-loaded microspheres was performed and two days after injection, tacrolimus plasma levels were again increased and both IL-2 plasma levels and PP2B activity decreased. A single subcutaneous tacrolimus-loaded microspheres injection was enough to reduce tacrolimus-related immunosuppressive parameters. These results open the possibility of new therapeutic strategies to administrate calcineurin inhibitors reducing the variability of their circulating levels related to gastrointestinal drug absorption/metabolism modifications., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background/aims: Kidneys from uncontrolled non heart-beating donors achieve a good level of renal function after transplantation. However, a number of them will never function in the recipient. Our aim was to determine if serum biomarkers associated with platelet activity, inflammation and the nitric oxide system in uncontrolled non heart-beating donors may help to predict no renal function recovery after renal transplantation., Methods: Serum levels of interleukin (IL)-6, IL-10, intercellular cell adhesion molecule-1 (ICAM-1), cyclic guanosine monophosphate (cGMP), nitrite + nitrate and platelet factor-4 (PF4) were measured using enzyme-linked immunosorbent assay (ELISA) kits in 88 uncontrolled non heart-beating donors divided according to the renal functionality achieved in the recipients into functional (n = 76) and non functional (n = 12)., Results: Kidneys from donors with higher IL-6 levels (>900 pg/ml) were functional after transplantation. Serum cGMP levels below 372.3 fmol/l were also associated with kidneys that recovered the renal function. However, serum levels of PF4 showed the best correlation with recovery of renal functional in the recipients since they were significantly lower in the donors whose kidneys functioned after transplantation., Conclusions: Serum PF4 levels in uncontrolled non heart-beating donors may be a good predictor for kidneys that never will reach functional recovery. Some serum cGMP, IL-6 and IL-10 levels may simply help identify kidneys that will function after transplantation.

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., (© 2015 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.)

It has been suggested that activated brown adipose tissue (BAT) shows increased glucose metabolic activity. However, less is known about metabolic activity of BAT under conditions of fasting and normal temperature. The aim of this study was to compare the possible differences in energetic metabolism between BAT and white adipose tissue (WAT) obtained from rabbits under the conditions of physiological temperature and 24 h after fasting conditions. The study was carried out on New Zealand rabbits (n=10) maintained for a period of 8 weeks at 23±2 °C. Food was removed 24 h before BAT and WAT were obtained. Protein expression levels of the glycolytic-related protein, glyceraldehyde-3-phosphate dehydrogenase, and pyruvate dehydrogenase were higher in WAT than that in BAT. The expression level of carnitine palmitoyltransferase 1 (CPT1) and CPT2, two fatty acid mitochondrial transporters, and the fatty acid β-oxidation-related enzyme, acyl CoA dehydrogenase, was higher in BAT than in WAT. Cytosolic malate dehydrogenase expression and malate dehydrogenase activity were higher in WAT than in BAT. However, lactate dehydrogenase expression and lactate content were significantly higher in BAT than in WAT. In summary, this study for the first time, to our knowledge, has described how under fasting and normal temperature conditions rabbit BAT seems to use anaerobic metabolism to provide energetic fuel, as opposed to WAT, where the malate-aspartate shuttle and, therefore, the gluconeogenic pathway seem to be potentiated., (© 2015 Society for Endocrinology.)

Aim: Further to its pivotal role in haemostasis, factor Xa (FXa) promotes effects on the vascular wall. The purpose of the study was to evaluate if FXa modifies the expression level of energy metabolism and oxidative stress-related proteins in femoral arteries obtained from type 2 diabetic patients with end-stage vasculopathy., Methods: Femoral arteries were obtained from 12 type 2 diabetic patients who underwent leg amputation. Segments from the femoral arteries were incubated in vitro alone and in the presence of 25 nmol l(-1) FXa and 25 nmol l(-1) FXa + 50 nmol l(-1) rivaroxaban., Results: In the femoral arteries, FXa increased triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase isotype 1 expression but decreased pyruvate dehydrogenase expression. These facts were accompanied by an increased content of acetyl-CoA. Aconitase activity was reduced in FXa-incubated femoral arteries as compared with control. Moreover, FXa increased the protein expression level of oxidative stress-related proteins which was accompanied by an increased malonyldialdehyde arterial content. The FXa inhibitor, rivaroxaban, failed to prevent the reduced expression of pyruvate dehydrogenase induced by FXa but reduced acetyl-CoA content and reverted the decreased aconitase activity observed with FXa alone. Rivaroxaban + FXa but not FXa alone increased the expression level of carnitine palmitoyltransferase I and II, two mitochondrial long chain fatty acid transporters. Rivaroxaban also prevented the increased expression of oxidative stress-related proteins induced by FXa alone., Conclusions: In femoral isolated arteries from type 2 diabetic patients with end-stage vasculopathy, FXa promoted disruption of the aerobic mitochondrial metabolism. Rivaroxaban prevented such effects and even seemed to favour long chain fatty acid transport into mitochondria., (© 2014 The British Pharmacological Society.)

Hydroxychloroquine has been shown to be efficacious in the treatment of autoimmune diseases, including systemic lupus erythematosus. Hydroxychloroquine-treated lupus patients showed a lower incidence of thromboembolic disease. Endothelial dysfunction, the earliest indicator of the development of cardiovascular disease, is present in lupus. Whether hydroxychloroquine improves endothelial function in lupus is not clear. The aim of this study was to analyze the effects of hydroxychloroquine on hypertension, endothelial dysfunction, and renal injury in a female mouse model of lupus. NZBWF1 (lupus) and NZW/LacJ (control) mice were treated with hydroxychloroquine 10 mg/kg per day by oral gavage, or with tempol and apocynin in the drinking water, for 5 weeks. Hydroxychloroquine treatment did not alter lupus disease activity (assessed by plasma double-stranded DNA autoantibodies) but prevented hypertension, cardiac and renal hypertrophy, proteinuria, and renal injury in lupus mice. Aortae from lupus mice showed reduced endothelium-dependent vasodilator responses to acetylcholine and enhanced contraction to phenylephrine, which were normalized by hydroxychloroquine or antioxidant treatments. No differences among all experimental groups were found in both the relaxant responses to acetylcholine and the contractile responses to phenylephrine in rings incubated with the nitric oxide synthase inhibitor N(G)-nitro-l-arginine methyl ester. Vascular reactive oxygen species content and mRNA levels of nicotinamide adenine dinucleotide phosphate oxidase subunits NOX-1 and p47(phox) were increased in lupus mice and reduced by hydroxychloroquine or antioxidants. Chronic hydroxychloroquine treatment reduced hypertension, endothelial dysfunction, and organ damage in severe lupus mice, despite the persistent elevation of anti-double-stranded DNA, suggesting the involvement of new additional mechanisms to improve cardiovascular complications., (© 2014 American Heart Association, Inc.)

Background: Several mechanisms have been proposed to explain why some platelets have a reduced response to aspirin (ASA). Among them, it was reported an increased circulating level of vitamin-D-binding protein (DBP). In addition, nitric oxide (NO) released from mononuclear cells was involved in the antiplatelet effects of ASA. The aim was to analyse the relationship between platelet response to ASA and both NO generation and vitamin-D-binding protein content in mononuclear cells., Materials and Methods: Mononuclear cells were obtained from patients with stable coronary artery disease that were divided by a platelet functionality test (PFA-100) as ASA-sensitive (n=23) and ASA resistant (n=27)., Results: Both the release of NO (determined by nitrite+nitrate concentration) and the expression of endothelial-type NO synthase (eNOS) were higher in mononuclear cells from ASA sensitive as compared with those from ASA-resistant patients. There was a positive correlation between either the release of NO and the expression of eNOS protein in mononuclear cells with the ability of ASA to inhibit platelet activity. DBP content in mononuclear cells was higher in ASA resistant than in ASA sensitive. The level of DBP content in mononuclear cells was negatively associated with the ability of ASA to inhibit platelets. However, in vitro experiments suggested that there was no association between DBP and NO production by mononuclear cells., Conclusions: Mononuclear cells from patients with platelets with lower responsiveness to ASA showed a reduced ability to produce NO., (© 2014 Stichting European Society for Clinical Investigation Journal Foundation.)

Objective: To analyse if platelet responsiveness to aspirin (ASA) may be associated with a different ability of platelets to generate nitric oxide (NO)., Patients/methods: Platelets were obtained from 50 patients with stable coronary ischemia and were divided into ASA-sensitive (n = 26) and ASA-resistant (n = 24) using a platelet functionality test (PFA-100)., Results: ASA-sensitive platelets tended to release more NO (determined as nitrite + nitrate) than ASA-resistant platelets but it did not reach statistical significance. Protein expression of nitric oxide synthase 3 (NOS3) was higher in ASA-sensitive than in ASA-resistant platelets but there were no differences in the platelet expression of nitric oxide synthase 2 (NOS2) isoform. The highest NOS3 expression in ASA-sensitive platelets was independent of the presence of T-to-C mutation at nucleotide position -786 (T(-786) → C) in the NOS3-coding gene. However, platelet content of phosphorylated NOS3 at Serine (Ser)(1177), an active form of NOS3, was higher in ASA-sensitive than in ASA-resistant platelets. The level of platelet NOS3 Ser(1177) phosphorylation was positively associated with the closure time in the PFA-100 test. In vitro, collagen failed to stimulate the aggregation of ASA-sensitive platelets, determined by lumiaggregometry, and it was associated with a significant increase (p = 0.018) of NOS3 phosphorylation at Ser(1177). On the contrary, collagen stimulated the aggregation of ASA-resistant platelets but did not significantly modify the platelet content of phosphorylated NOS3 Ser(1177). During collagen stimulation the release of NO from ASA-sensitive platelets was significantly enhanced but it was not modified in ASA-resistant platelets., Conclusions: Functional platelet responsiveness to ASA was associated with the platelet content of phosphorylated NOS3 at Ser(1177).

Introduction: Evidences have been suggested that phosphodiesterase type 5 (PDE5) inhibition promotes vasculoprotective benefits in patients with cardiovascular diseases., Aim: The aim of this study is to analyze the systemic effect of PDE5 inhibition in type 2 diabetes mellitus patients with erectile dysfunction (ED) determining changes in the expression levels of plasma proteins., Methods: Seventeen patients with controlled type 2 diabetes mellitus and ED were included in the study. Patients received vardenafil hydrochloride 20 mg on demand during 12 weeks. At the beginning and 12 weeks after vardenafil administration, plasma samples were collected and analyzed using proteomics., Main Outcome Measures: International Index of Erectile Function-Erectile Function Domain (IIEF-EFD) and plasma protein expression before and after vardenafil administration. Nitrate/nitrite release, PDE5, and soluble guanylate cyclase (sGC) expression and cyclic guanosine monophosphate (cGMP) content in cultured bovine aortic endothelial cells (BAECs)., Results: The IIEF-EFD score was markedly improved after 12 weeks of vardenafil administration. Plasma levels of alpha 1-antitrypsin isotypes 4 and 6 and β-tropomyosin were decreased, whereas apolipoprotein AI isoype 5 was increased 12 weeks after vardenafil administration. Only β-tropomyosin plasma levels were inversely correlated with IIEF-EFD score. Tropomyosin has been added to cultured BAECs and after 24 hours reduced the protein expression level of sGC-β1 subunit and decreased the cGMP content. Tropomyosin did not modify PDE5 expression and nitric oxide release in BAECs as compared with control BAECs. Vardenafil (10 μg/mL) did not modify sGC-β1 subunit expression in tropomyosin + vardenafil-incubated BAECs; however, vardenafil significantly reversed the reduction of cGMP content induced by tropomyosin., Conclusion: Vardenafil administration improved erectile functionality in controlled type 2 diabetes mellitus patients with ED, which was associated with reduction of circulating plasma β-tropomyosin levels. Tropomyosin affected by itself the cGMP generating system suggesting a possible new mechanism involved in ED. Vardenafil reversed the reduction effect of cGMP content elicited by tropomyosin in BAECs., (© 2013 International Society for Sexual Medicine.)

Background: Heart produces ATP through long-chain fatty acids beta oxidation., Purpose: To analyze whether in ventricular myocardium, high-fat diet may modify the expression of proteins associated with energy metabolism before myocardial function was affected., Methods: Wistar Kyoto rats were divided into two groups: (a) rats fed standard diet (control; n = 6) and (b) rats fed high-fat diet (HFD; n = 6). Proteins from left ventricles were analyzed by two-dimensional electrophoresis, mass spectrometry and Western blotting., Results: Rats fed with HFD showed higher body weight, insulin, glucose, leptin and total cholesterol plasma levels as compared with those fed with standard diet. However, myocardial functional parameters were not different between them. The protein expression of 3-ketoacyl-CoA thiolase, acyl-CoA hydrolase mitochondrial precursor and enoyl-CoA hydratase, three long-chain fatty acid β-oxidation-related enzymes, and carnitine-O-palmitoyltransferase I was significantly higher in left ventricles from HFD rats. Protein expression of triosephosphate isomerase was higher in left ventricles from HFD rats than in those from control. Two α/β-enolase isotypes and glyceraldehyde-3-phosphate isomerase were significantly increased in HFD rats as compared with control. Pyruvate and lactate contents were similar in HFD and control groups. Expression of proteins associated with Krebs cycle and mitochondrial oxidative phosphorylation was higher in HFD rats., Conclusions: Expression of proteins involved in left ventricle metabolic energy was enhanced before myocardial functionality was affected in rats fed with HFD. These findings may probably indicate higher cardiac energy requirement due to weight increase by HFD.

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 patient's 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 patient's 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.

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., (Copyright © 2012 Wiley Periodicals, Inc.)

The aim was to analyze the effect of tacrolimus on the aortic expression of proteins associated with the energetic metabolism and cytoskeleton and if it could be reverted by ET-1-receptor antagonist bosentan. Wistar Kyoto rats were divided into: control (n=10), tacrolimus (n=10, 0.5mg/kg bw/day tacrolimus for 30 days) and tacrolimus+bosentan (n=10, 0.5mg/kg bw/day tacrolimus and 100mg/kg bw/day bosentan for 30 days). Rat aortic segments were homogenized and submitted to 2-dimensional electrophoresis and mass spectrometry. Tacrolimus treatment did not modify neither systolic nor diastolic arterial pressure but increased ET-1 content, ET(A)- and ET(B)-type receptor expression in aorta. Proteomic study revealed that tacrolimus treatment modified the expression of aortic proteins associated with the cytoskeleton as some isotypes of lamin A and β-tropomyosin; and energetic metabolism such as ATP synthase gamma chain, NADH dehydrogenase ubiquinone, acyl CoA dehydrogenase long chain mitochondrial and phosphatidylinositol 3-kinase regulatory subunit gamma. Aortic expression of gp91-phox and MnSOD was also increased by tacrolimus. Bosentan co-administration with tacrolimus prevented also changes in ET-1 content and the expression of proteins associated with energetic metabolism. Bosentan did not affect the increased expression of gp91-phox related to tacrolimus although significantly enhanced aortic MnSOD expression. As conclusion, tacrolimus treatment increased ET-1 content in aortic wall and modified the expression of proteins associated with the cytoskeleton and energetic metabolism independently of changes on blood pressure. Bosentan reverted some effects induced by tacrolimus in the aorta and increased the antioxidant defense system., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Purpose: Although BRCA1 gene mutations have been associated with breast cancer, BRCA1 mutations have been also involved in other functions. Thrombosis and coagulation are novel mechanisms recently associated with cancer. The aims of the present study were (a) to evaluate, using proteomics, if BRCA1 mutation carriers have a different plasma proteins expression related to thrombosis and coagulation profile than non-mutant BRCA1 women and (b) to analyze if the expression of these proteins may be different among BRCA1 mutation carriers with and without breast cancer., Methods: Proteomic study was based on 2-dimensional electrophoresis and mass spectrometry. The study was performed in 10 BRCA1 non-mutant controls and 21 women with BRCA1 mutations (with breast cancer (n = 8) and breast cancer-free (n = 13)), all of them free of family history or diagnosis of ovarian cancer., Results: Proteomic study showed that fibrinogen gamma chain isotypes 2 and 3, serotransferrin isotype 4, and convertase C3/C5 isotypes 1-5 were significantly increased in plasma from BRCA1 mutation carriers with respect to BRCA1 non-mutant controls. Plasma levels of alpha-1 antitrypsin isotypes 2-5, apolipoprotein A-IV, and vitamin D-binding protein isotypes 1 and 2 were significantly reduced in BRCA1 mutation carriers with respect to non-mutant controls. Only apolipoprotein A-IV plasma levels were significantly higher in cancer-free BRCA1 mutations carriers compared with BRCA1 mutations carriers who developed breast cancer., Conclusion: It is suggested that independently of breast cancer generation, BRCA1-encoded gene alterations are associated with changes in the expression of circulating proteins associated with thrombosis and coagulation.

Stroke patients have a high risk of vascular recurrence. Biomarkers related to vascular recurrence, however, remain to be identified. The aim of the study was to identify, through proteomic analysis, plasma biomarkers associated with vascular recurrence within one year after the first ischemic stroke. This is a substudy (n = 134) of a large prospective multicenter study of post-stroke patients with an ischemic stroke. Plasma samples were obtained at inclusion. Among the identified proteins, only plasma levels of desmoplakin I were associated with protection against a new vascular event (Odds ratio: 0.64; 95% CI: 0.46-0.89; p = 0.009) after adjustment for hypercholesterolemia, statins and previous atherothrombotic stroke subtype. A greater number of patients without vascular recurrence had been treated with statins within three months of the recent ischemic stroke. Only patients who had been taking statins for 3 months after the ischemic stroke and did not suffer vascular recurrence over a follow-up year, have higher levels of desmoplakin I at the time of inclusion (Odds ratio 0.49; 95% CI: 0.28-0.86; p = 0.013). Increased desmoplakin I levels, determined within 1-3 months of the first ischemic stroke, could be a biomarker for statin responsiveness against a new vascular event in post-ischemic stroke patients taking statins early (1-3 months) after the ischemic stroke., (© 2012 The Authors. Journal of Neurochemistry © 2012 International Society for Neurochemistry.)

Objective: The purpose of this study was to evaluate the expression of proteins related to cytoskeleton and energetic metabolism at abdominal aortic aneurysm (AAA) sites using proteomics. Several remodeling-related mechanisms have been associated with AAA formation but less is known about the expression of proteins associated with cytoskeleton and energetic metabolism in AAAs., Methods: AAA samples (6.73 ± 0.40 cm size) were obtained from 13 patients during elective aneurysm repair. Control abdominal aortic samples were obtained from 12 organ donors. Proteins were analyzed using two-dimensional electrophoresis and mass spectrometry., Results: The expression of filamin was increased in the AAA site compared to control abdominal aortic samples while microfibril-associated glycoprotein-4 isotype 1, annexin A5 isotype 1, and annexin A2 were reduced compared with control abdominal aortic samples. Reduction in expression level of energetic metabolism-associated proteins such as triosephosphate isomerase, glyceraldehyde 3-phosphate dehydrogenase, and cytosolic aldehyde dehydrogenase was also observed in AAAs compared to controls. Reduction of triosephosphate isomerase expression was also observed by Western blot, which was accompanied by diminished triosephosphate isomerase activity. At the AAA site, pyruvate dehydrogenase expression was reduced and the content of both lactate and pyruvate was increased with respect to controls without changes in lactate dehydrogenase activity., Conclusions: The present results suggest that an anaerobic metabolic state may be favored further to reduce the expression of cytoskeleton-related proteins. The better knowledge of molecular mechanism involved in AAAs may favor development of new clinical strategies., (Copyright © 2012 Society for Vascular Surgery. Published by Mosby, Inc. All rights reserved.)

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.

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., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The purpose of the study was to determine if an intensive pre- season training program modifies the inflammatory status in professional soccer players and if this inflammatory profile may be associated with the physical state. We compared plasma protein biomarkers, using proteomics, and the physiological state and cardiac function in 12 professional soccer players and 9 recreational soccer players. Reduced cardiac low frequency [LF] after the pre- season training program previous competition with respect to recreational soccer players was found. No differences were found in cardiac high frequency, cardiac high frequency/low frequency ratio, tension index and oxygen volume consumption. Alpha-1-antitrypsin isotype-3, fibrinogen-gamma isotypes-1, 2 and 3 and vitamin-D-binding protein isotype-1 were reduced in professionals players compared with those in recreational players. However, an increased content of alpha-1-antitrypsin isotype-6 and alpha-1-antichymotrypsin 1 and 4 were found in professional soccer players. Spearman's analysis showed a positive correlation between LF and fibrinogen-gamma chain isotype 3; but LF was negatively correlated with alpha-antichymotrypsin isotype 4. Professional soccer players submitted to an intensive training showed differences in the content of plasma proteins associated with inflammatory/oxidative stress and thrombosis with respect to recreational soccer players. Proteomics analysis in combination with the analysis of cardiac function assessment may be useful to know more in depth molecular processes associated with sport and intensive exercise. Key pointsProteomics allow us to find differences in the plasma protein content in sportsmen.Just after pre-season training program, professional soccer players showed lower content of circulating proteins associated with inflammation compared to recreational soccer players.Proteomic analysis in combination with the analysis of cardiac function may be useful to know more in depth molecular inflammatory and oxidative processes associated with the sport.

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., (© 2010 Wiley-Liss, Inc.)

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)., Methods and Results: Samples from RAA and LAA were obtained from the same patient. Proteins were separated in 2-dimensional electrophoresis and identified by mass spectrometry. LAA from SR patients upexpressed alpha-actin isotype 1 and desmin isotypes 3 and 5 with respect to RAA. In LAA from AF patients were upexpressed cardiac alpha-actin isotypes 1 and 2, tropomyosin alpha- and beta-chains, and myosin light chain embryonic muscle/atrial isoform with respect to LAA from SR patients. In RAA from AF patients also upexpressed different cytoskeleton associated proteins with respect to RAA from SR patients. Different energetic metabolism-associated proteins were upexpressed in LAA and RAA from AF with respect those from SR patients. In AF patients, the expression of proteins associated with cardiac cytoprotection such as gluthatione-S-transferase, heat shock protein (Hsp) 27, and different Hsp60 isotypes, were higher in RAA but not in LAA with respect to the corresponding appendages in SR patients., Conclusions: For each individual patient RAA and LAA showed a similar level of proteins expressed associated with cytoskeleton, energetic metabolism, and cardiac cytoprotection. There were more differences in the level of proteins associated with the above-mentioned mechanisms between the atrial appendages from AF with respect to SR patients, which may open new targets for drugs.

Background: Different works have suggested that in the hypertrophied heart the energy metabolic pathway shifts to glycolysis. Our aim was to evaluate using proteomics the expression of proteins associated with different energetic metabolic pathways in hypertrophied left ventricles of spontaneously hypertensive rats (SHR)., Methods: 24-weeks-old SHR with stable hypertension and established left ventricle hypertrophy were used. Normotensive Wistar Kyoto rats were used as control. Proteins from left ventricles were analyzed by 2-dimensional electrophoresis and identified by comparison with a virtual rat heart proteomic map and mass spectrometry., Results: Enoyl-CoA hydratase expression, an enzyme involved in fatty acid beta-oxidation, was reduced whereas the expression of other beta-oxidation enzymes, 3-ketoacyl-CoA thiolase and the mitochondrial precursor of acyl-CoA thioester hydrolase, was increased in the hypertrophied left ventricles. The expression of two enzymes involved in the first steps of glycolysis, fructose bisphosphate aldolase and triosephosphate isomerase, was reduced in the left ventricle of SHR. Pyruvate dehydrogenase expression, enzyme involved in glucose oxidation, was enhanced in the hypertrophied ventricles whereas proteins of the tricarboxylic acid cycle were not modified. Proteins involved in the mitochondrial oxidative phosphorylation were overexpressed whereas the alpha-subunit of the mitochondrial precursor of ATP synthase was downexpressed., Conclusions: Several proteins involved in the main energy metabolic pathways were up and downexpressed. Moreover, our results seem to suggest that probably neither fatty acid beta-oxidation nor glycolysis are the only sources for energy in the hypertrophied left ventricle., (Copyright 2010 S. Karger AG, Basel.)

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.

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.

Although many uremic patients show platelet dysfunctionality, there are others with normal platelet functionality and even with thrombotic tendencies. Our aim was to evaluate changes in the expression of proteins in functional and dysfunctional uremic platelets. Using the platelet function analyzer (PFA-100) assay, uremic patients were divided according to their platelet functionality into normal (n=7) and dysfunctional (n=8). There were no significant differences in the number of circulating platelets and hematocrit and hemoglobin levels. Two-dimensional electrophoresis and mass spectrometry were used to determine and identify changes in protein expression. The closure time (CT) in the PFA-100 assay was significantly prolonged in the dysfunctional uremic platelets. In the dysfunctional platelets, actin-interacting protein-1 isotype 1 was down-regulated, while integrin IIb was up-regulated. Glutathione-S-transferase isotypes 1 and 2 and peroxiredoxin VI were up-regulated in the dysfunctional platelets. Pearson analysis showed a negative correlation between the platelet expression of integrin IIb and creatinine clearance. A positive correlation was found between creatinine clearance and glutathione-S-transferase isotype 2. Serum uric acid concentration was positively correlated with CT values and glutathione-S-transferase isotype 1. In conclusion, the analysis of the protein expression in uremic platelets with normal and dysfunctional activity revealed differences which may occur at the megakaryocyte level.

The effect of pravastatin on matrix metalloproteinase-9 (MMP-9) and the level of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 was studied in explants of human abdominal aortic aneurysm (AAA) obtained from 13 patients. The effect of pravastatin on the apoptotic status of human AAA explants was also examined. Total MMP-9 content did not differ in human AAA explants incubated in vitro in the presence or absence of pravastatin (10-6 mol/L) for 48 h. TIMP-1 levels were significantly increased in pravastatin-incubated AAA explants, but TIMP-2 production was not modified by pravastatin. Western blot experiments showed that, whereas Bax expression was increased in pravastatin-incubated AAA explants, the expression of Bcl-2 was not modified. On the other hand, the ratio of the expression of Bax to Bcl-2, an apoptotic index, was not modified by pravastatin. In the human AAA explants, the increase in Bax expression, but not the increase in TIMP-1 expression elicited by pravastatin, was reversed by L-mevalonate, a downstream HMG-CoA reductase metabolite, suggesting that the expression of Bax and TIMP-1 followed HMG-CoA reductase-dependent and -independent pathways, respectively. In conclusion, pravastatin increases both TIMP-1 and Bax expression in human AAA explants without changes in either MMP-9 activity or the apoptotic status.

Lipid-lowering strategies, particularly with statins, have been extremely useful in the prevention of cardiovascular disease. However, many patients who receive statin monotherapy do not achieve the desired cardiovascular benefits. Accumulation of cholesteryl esters within macrophages constitutes the hallmark of foam cells during atherogenesis. The action of acyl-coenzyme A (CoA): cholesterol acyltransferase (ACAT) leads to formation of cholesterol esters. There are two different ACAT isoforms: ACAT1 and ACAT2. A considerable interest to develop ACAT inhibitors has been emerging. This review has been focused on the current knowledge about a new ACAT inhibitor, F12511 or eflucimibe, and more particularly on its antiatherosclerotic properties.

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.

Background: The aim of the present study was to identify, using proteomics, changes in the expression of proteins and protein isoforms mainly associated with inflammation and vascular damage in the coronary sinus of patients undergoing off-pump coronary artery bypass (OPCAB) surgery., Methods: Plasma from the coronary sinus of 18 patients undergoing OPCAB was obtained before the ischemic procedure and after revascularization of the left anterior descending artery (LAD) with an internal thoracic artery graft. The mean LAD ischemic time was 11.1+/-0.7 min. For the proteomic study, two-dimensional gel electrophoresis was performed., Results: A significant decrease in the expression of three fibrinogen gamma-chain (FGC) isoforms, three vitamin D-binding protein (DBP) isoforms, six haptoglobin (HPT) isoforms, two apolipoprotein (apo)-AI isoforms, one ceruloplasmin (CP) isoform and apoAIV was found after revascularization. Before anastomosis, negative correlations between blood flow and FGC isoforms 2 and 3 and positive correlations between blood flow and apoAI isoform 5 were observed. After anastomosis, we observed positive correlations between haptoglobin isoform 3 and DBP isoform 2 and blood flow. Before anastomosis, positive correlation between DBP isoform 2 and troponin I was observed. After LAD grafting, positive correlations between troponin I and HPT isoform 6, CP isoform 1 and apoAI isoforms 2 and 4 were observed. After the procedure, positive correlations between creatine kinase-MB and coronary sinus expression of FGC isoforms 1, 2 and 3 and HPT isoforms 1 and 2 were also observed. conclusions: In blood from coronary sinus the expression of a number of proteins and protein isoforms associated with inflammation and vascular protection was modified after OPCAB.

Evidence suggests that PTHrP [PTH (parathyroid hormone)-related protein] can act as an inflammatory mediator in several pathological settings including cardiovascular disease. The aim of the present study was to determine whether PTHrP might be involved in human platelet activation. We used a turbidimetric method to determine platelet aggregation. The expression of PTH1R (PTH type 1 receptor) in human platelets was analysed by Western blot and flow cytometry analyses. PTHrP-(1-36) (10(-7) mol/l) by itself failed to modify the activation of platelets. However, it significantly enhanced ADP-induced platelet activation, and also increased the ability of other agonists (thrombin, collagen and arachidonic acid) to induce platelet aggregation. H89 (10(-6) mol/l) and 25 x 10(-6) mol/l Rp-cAMPS (adenosine 3',5'-cyclic monophosphorothioate Rp-isomer), two protein kinase A inhibitors, and 25 x 10(-9) mol/l bisindolylmaleimide I, a protein kinase C inhibitor, partially decreased the enhancing effect of PTHrP-(1-36) on ADP-induced platelet activation. Meanwhile, 10(-6) mol/l PTHrP-(7-34), a PTH1R antagonist, as well as 10(-5) mol/l PD098059, a MAPK (mitogen-activated protein kinase) inhibitor, or a farnesyltransferase inhibitor abolished this effect of PTHrP-(1-36). Moreover, 10(-7) mol/l PTHrP-(1-36) increased (2-fold over control) MAPK activation in human platelets. PTH1R was detected in platelets, and the number of platelets expressing it on their surface in patients during AMI (acute myocardial infarction) was not different from that in a group of patients with similar cardiovascular risk factors without AMI. Western blot analysis showed that total PTH1R protein levels were markedly higher in platelets from control than those from AMI patients. PTH1R was found in plasma, where its levels were increased in AMI patients compared with controls. In conclusion, human platelets express the PTH1R. PTHrP can interact with this receptor to enhance human platelet activation induced by several agonists through a MAPK-dependent mechanism.

Our aim was to analyze the plasma proteome in aspirin (acetylsalicylic acid [ASA])-sensitive and ASA-resistant coronary ischemic patients. Plasma from 19 ASA-sensitive and 19 ASA-resistant patients was analyzed. For the proteomic study, two-dimensional electrophoresis was performed. The expression of one isotype of the fibrinogen gamma chain and three isotypes of haptoglobin was increased in ASA-resistant patients. Three vitamin D binding protein isotypes were increased in ASA-resistant patients. In vitro incubation of vitamin D binding protein (DBP) with blood from healthy volunteers reduced the inhibitory effect of ASA on thromboxane A2 production. DBP may be a new regulator of the inhibitory effect of ASA on platelets.

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) beta1-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 beta1-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 beta1-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.

F12511(S)-2',3',5'-trimethyl-4'-hydroxy-alpha-dodecylthio-alpha-phenylacetanilide (F12511) is a new Acyl-CoA cholesterol acyltransferase (ACAT) inhibitor that not only reduces the plasma cholesterol levels but also has anti-atherosclerotic actions in animals models. The study's aim was to analyze if F12511 may directly modify the ability of tumor necrosis factor--alpha (TNF-alpha)-incubated bovine aortic endothelial cells (BAEC) to express endothelial nitric oxide synthase (eNOS) protein and inflammatory-related proteins such as platelet endothelial cell adhesion molecule (PECAM) and CD40 ligand (CD40L). The addition of increasing concentrations of F12511 (10 to 10 mol/L) failed to modify the level of eNOS protein expressed in control BAEC. TNF-alpha (10 ng/mL) reduced the expression of eNOS protein. In TNF-alpha--incubated BAEC, F12511 protected eNOS expression in a concentration-dependent manner. TNF-alpha stimulated the expression of both CD40L and PECAM in cultured BAEC. F12511 (10 mol/L) failed to modify the expression of CD40L and PECAM in control and TNF-alpha-incubated BAEC. Reverse transcriptase polymerase chain reaction showed a marked expression of the ACAT-2 isoform and absent of expression of the ACAT-1 isoform in BAEC. The presence of ACAT-2 isoform in BAEC was further confirmed by Western blot. F12511 failed to modify the expression of the proinflammatory associated proteins PECAM and CD40L in the endothelium but protected eNOS expression in the endothelial cells exposed to inflammatory conditions.

Lead exposure induces dysfunction of the cyclic guanosine monophosphate-dependent vasodilator system through downregulation of soluble guanylate cyclase (sGC) expression. The endothelium not only releases vasodilators but also vasoconstrictors such as endothelin-1 (ET-1). Our aim was to explore the role of the vascular endothelium and ET-1 as possible mediators of lead-induced downregulation of sGC. Isolated aortic segments from Wistar Kyoto rats were incubated in the presence or absence of lead (1 parts per million) for 24 h. Endothelium was mechanically removed in some of the aorta segments. As reported previously, lead exposure induced downregulation of sGC protein expression in the intact aortic segments. However, lead exposure failed to significantly modify sGC-beta1 subunit expression in the endothelium-denuded aortic segments. Incubation with a selective ETA-type receptor inhibitor, BQ-123 (10(-6) mol/l), restored sGC protein expression in lead-exposed intact aortic segments. As it has also been previously observed, incubation in lead-containing medium resulted in the upregulation of cyclooxygenase-2 (COX-2) in the intact aortic segments. Denudation of endothelium partially abrogated this effect of lead. Incubation with BQ-123 prevented the lead-induced upregulation COX-2 in the intact aortic segments. However, neither ET-1 content nor ETA-type receptor expression were modified by lead exposure of the aortic segments. As conclusion, the endothelium through the activation of ETA-type receptors mediates the downregulation of sGC expression by lead in the vascular wall.

The aim of the present study was to use proteomics to analyse modifications in the level of expression of different proteins in BVSMCs (bovine vascular smooth muscle cells) incubated in the absence and presence of 17beta-oestradiol. By using two-dimensional electrophoresis with a pH range of 4-7, we identified several areas on the gels in which the level of expression of proteins were different between control BVSMCs and cells incubated for 24 h with 17beta-oestradiol. Changes in several isoforms of alpha-enolase, HSP60 (heat-shock protein 60), vimentin and PDI (protein disulphide-isomerase) were observed in BVSMCs. The expression of alpha-enolase isoform 1 was enhanced after 17beta-oestradiol treatment. The expression of HSP60 isoform 3, vimentin isoforms 2 and 3 and caldesmon was reduced by 17beta-oestradiol. Finally, the expression of PDI isoforms was reduced by 17beta-oestradiol. In summary, 17beta-oestradiol modified the expression of isoforms of proteins associated with smooth muscle cell proliferation (alpha-enolase, vimentin and HSP-60), cell contraction (vimentin and caldesmon) and cell redox modulation (PDI). These findings confirm that 17beta-oestradiol may modulate a wide range of signalling pathways in vascular smooth muscle cells.

Our aim was to analyse endothelial hypoxic preconditioning after hypoxia-reperfusion (HR). Endothelial functionality was analysed through the vasorelaxation responses to acetylcholine (Ach) and the level of serine1177 phosphorylated endothelial nitric oxide synthase (eNOS) (ser1177-eNOS) measured by Western blot in in vitro hypoxic preconditioned (P + HR) isolated rat aortic segments. Relaxation in response to Ach was reduced in phenylephrine-precontracted aortic segments after HR (control: IC50, 5 +/- 2.5 x 10(-8) mol l(-1); HR: IC50, 3 +/- 1.2 x 10(-7) mol l(-1); P < 0.05). Ach-dependent vasodilatation was improved by P + HR. The content of ser1177-eNOS in the HR segments was 1.5-fold lower than in P + HR. Confocal microscopy showed an increased content of both superoxide anion and peroxynitrite in the vascular wall of HR aortic segments, which it was reduced by P + HR. Geldanamycin (10 microg ml(-1)), an agent known to inhibit heat shock protein 90 (hsp90), reduced the level of ser1177-eNOS in P + HR aortic segments. However in the presence of geldanamycin, endothelial hypoxic preconditioning persisted. We conclude that short periods of hypoxia induced endothelial hypoxic preconditioning that was accompanied by enhanced levels of ser1177-eNOS in the vascular wall. The fact that endothelial hypoxic preconditioning persisted in the presence of geldanamycin suggests that other molecular mechanisms are involved in the endothelial adaptation to HR injury.

Background: The impaired renal function and vasodilatation that accompany age need to be re-addressed based upon the new knowledge concerning vascular nitric oxide (NO)-dependent systems. The present study examined the effects of age on the NO-related renal response., Methods: The study was performed in euvolaemic, conscious Wistar rats, aged 5 and 18 months. Renal function and haemodynamic measurements with fluorescent microspheres were employed to assess differences between groups., Results: A first set of experiments showed that ageing rats had a reduced natriuretic and diuretic response to acetylcholine, whereas the response to sodium nitroprusside was preserved. In the same regard, a reduction of the renal functional effects of L-arginine (L-Arg) and L-glycine (L-Gly) was found in the older rats. In the ageing rats, these responses were accompanied by an enhanced effect of the L-Arg competitive analogue, NwNLA, which provoked a marked reduction of renal function. This effect of NwNLA was blocked by the simultaneous administration of a small dose of L-Arg in the ageing but not in the young rats. Systemic haemodynamic studies revealed that in ageing rats, NwNLA reduced renal blood flow and increased renal vascular resistances in a significantly higher proportion than in younger animals. However, flow to other organs, namely, brain, spleen or liver, was affected in a similar manner in both young and old rats. Ultrastructural alterations were found in endothelial cells, which might constitute the anatomical basis for the observed functional derangements., Conclusions: The present experiments reveal that ageing is accompanied by significant differences in NO-related responses in the kidney which do not appear to affect blood flow to other organs. The response to L-Arg and L-Arg competitive analogues supports the existence of a marked dependency on NO-related mechanisms in the ageing rats, but not of a decreased baseline activity of the NO-dependent pathways.