Spike Protein Subunits of SARS-CoV-2 Alter Mitochondrial Metabolism in Human Pulmonary Microvascular Endothelial Cells: Involvement of Factor Xa.

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 µg/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.

Clinical and Surgical Outcomes in Extensive Scalp Reconstruction after Oncologic Resection: A Comparison of Anterolateral Thigh, Latissimus Dorsi and Omental Free Flaps.

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.

Platelet Apoptotic Response May Be Associated With the Capacity of Aspirin to Inhibit Platelets.

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.

Reduction from 2011 to 2017 in adherence to breast cancer screening and non-improvement in the uptake of cervical cancer screening among women living in Spain.

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/m2; 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/m2; 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.

Relationship Between Erectile Dysfunction, Diabetes and Dyslipidemia in Hypertensive-Treated Men.

PURPOSE: PRESIDEN study is a large study to analyze the erectile dysfunction (ED) incidence in Spanish population. The present study is a pilot sub-analysis from PRESIDEN to determine if ED or plasma testosterone (TST) level in controlled hypertensive patients may be associated with comorbidities and/or plasma nitrite+nitrate and antioxidant capacity. MATERIALS AND METHODS: Forty-four hypertensive individuals were aleatory selected from PRESIDEN study, matching by age (28 showing ED and 16 without ED). RESULT: Diabetes was present in 28.57% of ED patients and in 18.75% of patients without ED. In patients with and without ED, increasing age showed tendency of higher frequency of an additional comorbidity (diabetes or dyslipemia) (P = .09). Apparently, plasma TST levels were lower in older ED patients compared to younger patients with and without ED, although it did not reach statistical significance (P = .69). Older ED patients also showed lower TST levels than older patients without ED, although it was not statistical significant (16.15 ± 2.84 vs 13.91± 2.77; P = .69). Dyslipidemia was showed by 52.17% with lower TST (? nmol/L) while 23.80% of patients with plasma TST levels > 15 nmol/L had dyslipidemia. The percentage of ED patients was similar between patients with low and high TST levels. CONCLUSION: More ED hypertensive patients seem to show two comorbidities (diabetes and dyslipidemia) than hypertensivepatients without ED. Younger patients with ED tended to show more commonly diabetes than older ED patients. Plasma TST levels were not associated with more prevalence of ED but lower plasma TST levels showed tendency to higher prevalence of dyslipidemia.

New strategy of tacrolimus administration in animal model based on tacrolimus-loaded microspheres.

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.

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 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.

KCNH2 gene mutation: a potential link between epilepsy and long QT-2 syndrome.

Long QT syndrome (LQTS) is closely associated with syncope, seizure, and sudden death but LQTS is frequently misdiagnosed as epilepsy. LQTS and epilepsy both belong to the group of ion channelopathies that manifest in the heart and brain. Therefore, genetic analysis of genes associated with potassium and sodium homeostasis and electrical disorders may reveal a link between epilepsy and lethal cardiac arrhythmia. Here, the authors report a young woman who suffered recurrent seizure episodes and syncopes that occurred while walking and also during rest. She showed electroencephalogram abnormalities and a pathological prolonged QTc interval in electrocardiogram. The patient and the 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.

Changes by tacrolimus of the rat aortic proteome: involvement of endothelin-1.

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 ß-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.

Changes in the expression of plasma proteins associated with thrombosis in BRCA1 mutation carriers.

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.

Plasma desmoplakin I biomarker of vascular recurrence after ischemic stroke.

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.

Expression of cytoskeleton and energetic metabolism-related proteins at human abdominal aortic aneurysm sites.

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.

New and old mechanisms associated with hypertension in the elderly.

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