Analysis of the gut microbiota profile targeted to multiple hypervariable regions of 16S rRNA in a hypertensive heart failure rat model.

The gut microbiota, comprising a diverse community of microorganisms, significantly influences various aspects of health. Changes in the composition of the gut microbiota are implicated in adverse effects on host physiology, contributing to the pathogenesis of cardiovascular diseases, among others pathological conditions. Understanding the role of the gut microbiota in the context of heart failure is particularly important. In this regard, the spontaneously hypertensive heart failure (SHHF) rat is an adequate experimental model since exhibits many features in common with heart failure (HF) in humans. Recent advancements in next-generation sequencing (NGS) have greatly improved microbiome analysis. However, standardization and the adoption of best practices are essential to mitigate experimental variations across studies. This manuscript outlines a straightforward methodology for analyzing gut microbiota composition in SHHF rat fecal samples using 16S rRNA sequencing, emphasizing the relevance of gut microbiota in heart failure.

Special Issue: Gut Microbiota in Disease and Health 2.0.

In recent years, research on the gut microbiota has undeniably captivated the attention of students, investigators, clinicians, and the general public [...].

Gut Microbiota and Derived Short-Chain Fatty Acids Are Linked to Evolution of Heart Failure Patients.

There is a lack of direct evidence regarding gut microbiota dysbiosis and changes in short-chain fatty acids (SCFAs) in heart failure (HF) patients. We sought to assess any association between gut microbiota composition, SCFA production, clinical parameters, and the inflammatory profile in a cohort of newly diagnosed HF patients. In this longitudinal prospective study, we enrolled eighteen newly diagnosed HF patients. At admission and after 12 months, blood samples were collected for the assessment of proinflammatory cytokines, monocyte populations, and endothelial dysfunction, and stool samples were collected for analysis of gut microbiota composition and quantification of SCFAs. Twelve months after the initial HF episode, patients demonstrated improved clinical parameters and reduced inflammatory state and endothelial dysfunction. This favorable evolution was associated with a reversal of microbiota dysbiosis, consisting of the increment of health-related bacteria, such as genus Bifidobacterium, and levels of SCFAs, mainly butyrate. Furthermore, there was a decrease in the abundance of pathogenic bacteria. In vitro, fecal samples collected after 12 months of follow-up exhibited lower inflammation than samples collected at admission. In conclusion, the favorable progression of HF patients after the initial episode was linked to the reversal of gut microbiota dysbiosis and increased SCFA production, particularly butyrate. Whether restoring butyrate levels or promoting the growth of butyrate-producing bacteria could serve as a complementary treatment for these patients deserves further studies.

Gut Microbiota Dysbiosis in COVID-19: Modulation and Approaches for Prevention and Therapy.

Inflammation and oxidative stress are critical underlying mechanisms associated with COVID-19 that contribute to the complications and clinical deterioration of patients. Additionally, COVID-19 has the potential to alter the composition of patients' gut microbiota, characterized by a decreased abundance of bacteria with probiotic effects. Interestingly, certain strains of these bacteria produce metabolites that can target the S protein of other coronaviruses, thereby preventing their transmission and harmful effects. At the same time, the presence of gut dysbiosis can exacerbate inflammation and oxidative stress, creating a vicious cycle that perpetuates the disease. Furthermore, it is widely recognized that the gut microbiota can metabolize various foods and drugs, producing by-products that may have either beneficial or detrimental effects. In this regard, a decrease in short-chain fatty acid (SCFA), such as acetate, propionate, and butyrate, can influence the overall inflammatory and oxidative state, affecting the prevention, treatment, or worsening of COVID-19. This review aims to explore the current evidence regarding gut dysbiosis in patients with COVID-19, its association with inflammation and oxidative stress, the molecular mechanisms involved, and the potential of gut microbiota modulation in preventing and treating SARS-CoV-2 infection. Given that gut microbiota has demonstrated high adaptability, exploring ways and strategies to maintain good intestinal health, as well as an appropriate diversity and composition of the gut microbiome, becomes crucial in the battle against COVID-19.

Non-invasive electromechanical assessment during atrial fibrillation identifies underlying atrial myopathy alterations with early prognostic value.

Electromechanical characterization during atrial fibrillation (AF) remains a significant gap in the understanding of AF-related atrial myopathy. This study reports mechanistic insights into the electromechanical remodeling process associated with AF progression and further demonstrates its prognostic value in the clinic. In pigs, sequential electromechanical assessment during AF progression shows a progressive decrease in mechanical activity and early dissociation from its electrical counterpart. Atrial tissue samples from animals with AF reveal an abnormal increase in cardiomyocytes death and alterations in calcium handling proteins. High-throughput quantitative proteomics and immunoblotting analyses at different stages of AF progression identify downregulation of contractile proteins and progressive increase in atrial fibrosis. Moreover, advanced optical mapping techniques, applied to whole heart preparations during AF, demonstrate that AF-related remodeling decreases the frequency threshold for dissociation between transmembrane voltage signals and intracellular calcium transients compared to healthy controls. Single cell simulations of human atrial cardiomyocytes also confirm the experimental results. In patients, non-invasive assessment of the atrial electromechanical relationship further demonstrate that atrial electromechanical dissociation is an early prognostic indicator for acute and long-term rhythm control.

Relación entre la cuantificación de calcio coronario y la composición de la microbiota intestinal en sujetos sin enfermedad cardiovascular previa: estudio piloto / Relationship between the coronary artery calcium quantification and gut microbiota composition in subjects without previous cardiovascular disease: A pilot study

Objetivo: Determinar si existe alguna asociación entre el perfil de microbiota intestinal y la carga aterosclerótica global medida mediante cuantificación de calcio coronario (CCC) en sujetos sin antecedentes de enfermedad cardiovascular (ECV). Métodos: Se incluyeron 20 pacientes mayores de edad, sin antecedentes de ECV a los que se les hizo la CCC mediante tomografía computarizada multicorte. Además, se les recogió una muestra de heces para caracterizar la composición de la microbiota intestinal mediante la secuenciación del gen 16S RNAr con técnicas de secuenciación masiva y una muestra de sangre para la cuantificación de citoquinas proinflamatorias, mediante ELISA específicos, y del metabolito bacteriano N-óxido de trimetilamina (TMAO) por cromatografía de gases/líquidos acoplada a espectrometría de masas en tándem. Resultados: La media de edad fue de 63,5 años y un 60% eran mujeres. La mitad de los pacientes (n=10) presentaron una CCC>100 y se caracterizaron por una mayor abundancia de bacterias del filo Proteobacteria, pertenecientes principalmente a las familias Enterobacteriaceae y Pasteurellaceae, que los pacientes con CCC≤100. La mayoría de los géneros bacterianos identificados, Enterobacter, Escherichia/Shigella, Klebsiella, Citrobacter y Salmonella, se asociaron positivamente con los niveles plasmáticos de TNF-α o IL-1β y con la producción de TMAO. Conclusión: Los resultados de este estudio piloto muestran un perfil de microbiota intestinal asociado a la presencia de CCC>100 en pacientes sin ECV previa, caracterizado por un aumento en la proporción de géneros bacterianos productores de TMAO. Puesto que la composición de la microbiota intestinal es altamente modulable por diversos factores, es posible que, en un futuro, podamos prevenir, e incluso intervenir, la enfermedad cardiovascular mediante estrategias nutricionales. (AU)

Aim: To investigate the relationship between gut microbiota composition and the presence of coronary atherosclerosis assessed by coronary artery calcium (CAC) quantification in individuals without previous cardiovascular disease (CVD). Methods: We included 20 patients over 18 years of age with no history of CVD who underwent multiple detector-computed tomography. From each patient, a stool sample was obtained to characterize gut microbiota composition by sequencing bacterial 16S ribosomal RNA gene. In addition, circulating levels of TNF-α and IL-1β, as well as trimethylamine N-oxide (TMAO) were determined in plasma samples by automated ELISA and capillary gas chromatography-mass spectrometry, respectively. Results: The mean age of patients was 63.5 years and 60% were women. Half of patients had CAC >100 (Agatston score), and were characterized by a higher abundance of the phylum Proteobacteria, mainly of bacteria belonging to the families Enterobacteriaceae and than patients with a CAC ≤ 100. Moreover, bacterial genera identified as biomarkers, such as Enterobacter, Escherichia/Shigella y Klebsiella, were positively associated with inflammation levels and with TMAO production. Conclusion: Our data shows a gut microbiota profile associated with the presence of coronary calcium in patients without previous CVD. Although there are no strategies to decrease the amount of coronary calcium, gut microbiota is highly malleable by several factors. The possibility of preventing and even intervening CVD progression through strategies targeted gut microbiota is a very attractive idea that deserves further studies. (AU)

Relationship between the coronary artery calcium quantification and gut microbiota composition in subjects without previous cardiovascular disease: A pilot study. / Relación entre la cuantificación de calcio coronario y la composición de la microbiota intestinal en sujetos sin enfermedad cardiovascular previa: estudio piloto.

AIM: To investigate the relationship between gut microbiota composition and the presence of coronary atherosclerosis assessed by coronary artery calcium (CAC) quantification in individuals without previous cardiovascular disease (CVD). METHODS: We included 20 patients over 18 years of age with no history of CVD who underwent multiple detector-computed tomography. From each patient, a stool sample was obtained to characterize gut microbiota composition by sequencing bacterial 16S ribosomal RNA gene. In addition, circulating levels of TNF-α and IL-1ß, as well as trimethylamine N-oxide (TMAO) were determined in plasma samples by automated ELISA and capillary gas chromatography-mass spectrometry, respectively. RESULTS: The mean age of patients was 63.5 years and 60% were women. Half of patients had CAC >100 (Agatston score), and were characterized by a higher abundance of the phylum Proteobacteria, mainly of bacteria belonging to the families Enterobacteriaceae and than patients with a CAC ≤ 100. Moreover, bacterial genera identified as biomarkers, such as Enterobacter, Escherichia/Shigella y Klebsiella, were positively associated with inflammation levels and with TMAO production. CONCLUSION: Our data shows a gut microbiota profile associated with the presence of coronary calcium in patients without previous CVD. Although there are no strategies to decrease the amount of coronary calcium, gut microbiota is highly malleable by several factors. The possibility of preventing and even intervening CVD progression through strategies targeted gut microbiota is a very attractive idea that deserves further studies.

Identification of a Plasma Microrna Signature as Biomarker of Subaneurysmal Aortic Dilation in Patients with High Cardiovascular Risk.

Patients with subaneurysmal aortic dilation (SAD; 25-29 mm diameter) are likely to progress to true abdominal aortic aneurysm (AAA). Despite these patients having a higher risk of all-cause mortality than subjects with aortic size <24 mm, early diagnostic biomarkers are lacking. MicroRNAs (miRs) are well-recognized potential biomarkers due to their differential expression in different tissues and their stability in blood. We have investigated whether a plasma miRs profile could identify the presence of SAD in high cardiovascular risk patients. Using qRT-PCR arrays in plasma samples, we determined miRs differentially expressed between SAD patients and patients with normal aortic diameter. We then selected 12 miRs to be investigated as biomarkers by construction of ROC curves. A total of 82 significantly differentially expressed miRs were found by qPCR array, and 12 were validated by qRT-PCR. ROC curve analyses showed that seven selected miRs (miR-28-3p, miR-29a-3p, miR-93-3p, miR-150-5p, miR-338-3p, miR-339-3p, and miR-378a-3p) could be valuable biomarkers for distinguishing SAD patients. MiR-339-3p showed the best sensitivity and specificity, even after combination with other miRs. Decreased miR-339-3p expression was associated with increased aortic abdominal diameter. MiR-339-3p, alone or in combination with other miRs, could be used for SAD screening in high cardiovascular risk patients, helping to the early diagnosis of asymptomatic AAA.

Gut Microbiota Profile Identifies Transition From Compensated Cardiac Hypertrophy to Heart Failure in Hypertensive Rats.

Microcirculatory alterations displayed by patients with heart failure (HF) induce structural and functional intestinal changes that may affect normal gut microbial community. At the same time, gut microbiota can influence pathological mechanisms implicated in HF progression. However, it is unknown whether gut microbiota dysbiosis can precede the development of cardiac alterations in HF or it is only a mere consequence. Our aim was to investigate the potential relationship between gut microbiota composition and HF development by comparing spontaneously hypertensive heart failure and spontaneously hypertensive rat models. Gut microbiota from spontaneously hypertensive heart failure, spontaneously hypertensive rat, and normotensive Wistar Kyoto rats at 9 and 19 months of age was analyzed by sequencing the 16S ribosomal RNA gene, and KEGG metabolic pathways associated to 16S profiles were predicted. Beta diversity, Firmicutes/Bacteroidetes ratio, taxonomic abundances, and potential metabolic functions of gut microbiota were significantly different in spontaneously hypertensive heart failure with respect to spontaneously hypertensive rat before (9 months) and after (19 months) cardiac differences were presented. Nine-month-old spontaneously hypertensive heart failure showed a significant increase in the genera Paraprevotella, Oscillospira, Prevotella 9, Faecalitalea, Faecalibacterium, Ruminiclostridium 6, Phascolarctobacterium, Butyrivibrio, Parasutterella, and Parabacteroides compared with both Wistar Kyoto and spontaneously hypertensive rat, while Ruminiclostridium 9, Oscillibacter, Ruminiclostridium, Mucispirillum, Intestinimonas, and Akkermansia were diminished. Of them, Akkermansia, Prevotella 9, Paraprevotella, and Phascolarctobaterium were associated to changes in cardiac structure and function. Our results demonstrate an association between specific changes in gut microbiota and the development of HF in a hypertensive model of HF and further support the intervention to restore gut microbiota as an innovative therapeutic strategy for preventing HF.

Effect of intensive multifactorial treatment on vascular progenitor cells in hypertensive patients.

BACKGROUND: Most hypertensive patients, despite a proper control of their cardiovascular risk factors, have cardiovascular complications, evidencing the importance of controlling and/or reversing target-organ damage. In this sense, endothelial dysfunction has been associated with the presence of cardiovascular risk factors and related cardiovascular outcomes. Since hypertension often clusters with other risk factors such as dyslipemia, diabetes and obesity, in this study we have investigated the effect of intensive multifactorial treatment on circulating vascular progenitor cell levels on high-risk hypertensive patients. DESIGN: We included108 hypertensive patients receiving intensive multifactorial pharmacologic treatment and dietary recommendations targeting blood pressure, dyslipemia, hyperglycemia and weight for 12 months. After the treatment period, blood samples were collected and circulating levels of endothelial (CD34+/KDR+, CD34+/VE-cadherin+) and smooth muscle (CD14+/endoglin+) progenitor cells were identified by flow cytometry. Additionally, plasma concentration of vascular endothelial growth factor (VEGF) was determined by ELISA. RESULTS: Most hypertensive patients (61±12 years, 47% men) showed cardiovascular parameters within normal ranges at baseline. Moreover, body mass index and the majority of the biochemical parameters (systolic and diastolic blood pressure, fasting glucose, total cholesterol, HDL-c, LDL-c, creatinine and hs-CRP) significantly decreased overtime. After 12 months of intensive treatment, CD34+/KDR+ and CD14+/endoglin+ levels did not change, but CD34+/VE-cadherin+ cells increased significantly at month 12 [0.9(0.05-0.14)% vs 0.05(0.02-0.09)% P<0.05]. However, VEGF plasma concentration decreased significantly overtime [89.1(53.9-218.7) vs [66.2(47.5-104.6) pg/mL, P<0.05]. CONCLUSIONS: Long-term intensive treatment in hypertensive patients further improves cardiovascular risk and increases circulating EPCs, suggesting that these cells could be a therapeutic target.

BRCA2 gene mutations and coagulation-associated biomarkers.

Thromboembolic events are the second cause of death in cancer patients, although the mechanisms underlying this increased thromboembolic risk remain unclear. The aims of this study were to examine whether BRCA2 gene mutations may modify the circulating levels of thrombocoagulation biomarkers and whether breast cancer development may influence changes in such circulating biomarkers. The study was performed in 25 women with mutations in the BRCA2 gene (n=12 breast cancer, n=13 breast cancer-free) and in 13 BRCA2 non-mutant controls. Results revealed that plasma levels of fibrinogen gamma chain isotypes 2 and 3, haptoglobin isotypes 4 and 5, serotransferrin isotypes 3 and 4 and convertase C3/C5 isotypes 4 and 5 were significantly higher in BRCA2 mutation carriers compared to controls. However, plasma levels of vitamin D binding protein isotype 1 and alpha1-antitrypsin isotypes 2, 3 and 4 were significantly decreased in BRCA2 mutation carriers compared to controls. Plasma expression of PF4 and P-selectin was significantly higher in BRCA2 mutations carriers than in controls. BRCA2 truncated mutations conserving a binding region for RAD51 were associated with increased plasma levels of alpha1-antitrypsin isotypes 3 and 4 with respect to women showing BRCA2 mutations that loss the binding RD51 region to BRCA2. Only plasma levels of vitamin D binding protein isotypes 1 and 3 were significantly reduced and alpha 1-antitrypsin isotype 1 was increased in cancer-free BRCA2 mutation carriers compared to BRCA2 mutation carriers with breast cancer. The presence of BRCA2 mutations is associated with increased plasma levels of thrombo-coagulating-related proteins, which are independent to breast cancer development.

Serum biomarkers in uncontrolled no heart-beating donors may identify kidneys that will never work after transplantation.

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.

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 ß-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 hormones on platelet aggregation.

Platelets and their activation/inhibition mechanisms play a central role in haemostasis. It is well known agonists and antagonists of platelet activation; however, during the last years novel evidences of hormone effects on platelet activation have been reported. Platelet functionality may be modulated by the interaction between different hormones and their platelet receptors, contributing to sex differences in platelet function and even in platelet-mediated vascular damage. It has suggested aspects that apparently are well established should be reviewed. Hormones effects on platelet activity are included among them. This article tries to review knowledge about the involvement of hormones in platelet biology and activity.

Effects of factor Xa on the expression of proteins in femoral arteries from type 2 diabetic patients.

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.

Nitric oxide from mononuclear cells may be involved in platelet responsiveness to aspirin.

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.

Platelet content of nitric oxide synthase 3 phosphorylated at Serine 1177 is associated with the functional response of platelets to aspirin.

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).

Vardenafil improves penile erection in type 2 diabetes mellitus patients with erectile dysfunction: role of tropomyosin.

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 ß-tropomyosin were decreased, whereas apolipoprotein AI isoype 5 was increased 12 weeks after vardenafil administration. Only ß-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-ß1 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-ß1 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 ß-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.

Changes in cardiac energy metabolic pathways in overweighed rats fed a high-fat diet.

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 ß-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/ß-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.

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.