Impact of treatment on myocardial lysyl oxidase expression and collagen cross-linking in patients with heart failure

The aim of this study was to investigate whether torasemide modifies collagen cross-linking in the failing human heart. We analyzed the degree of cross-linking and the expression of the enzyme lysyl oxidase, which regulates cross-linking, in the myocardium of patients with chronic heart failure at baseline and after 8 months of treatment with either torasemide or furosemide in addition to their standard heart failure therapy. Whereas lysyl oxidase protein expression was very scarce in normal hearts, it was highly expressed in failing hearts. Cross-linking was increased (P<0.001) in heart failure patients compared with normal hearts. These 2 parameters decreased (P=0.021 and P=0.034) in torasemide-treated patients and remained unchanged in furosemide-treated patients. In addition, more (P=0.009) patients showed normalization of left ventricular chamber stiffness in the torasemide subgroup than in the furosemide subgroup after treatment. Lysyl oxidase expression correlated with cross-linking (r=0.661; P<0.001), and cross-linking correlated with left ventricular chamber stiffness (r=0.452; P=0.002) in all patients. These findings show for the first time that lysyl oxidase overexpression is associated with enhanced collagen cross-linking in the failing human heart. In addition, we report that the ability of torasemide to correct both lysyl oxidase overexpression and enhanced collagen cross-linking results in normalization of left ventricular chamber stiffness in patients with heart failure. Lysyl oxidase may thus represent a target for reduction of stiff collagen and improvement of left ventricular mechanical properties in heart failure patients

Polymorphisms and promoter overactivity of the p22(phox) gene in vascular smooth muscle cells from spontaneously hypertensive rats

In a previous study, we found that the p22(phox) subunit of the NADH/NADPH oxidase is overexpressed in vascular smooth muscle cells (VSMCs) from spontaneously hypertensive rats (SHRs) with enhanced vascular production of superoxide anion ((.)O(2)(-)). Thus, we have investigated whether changes in the sequence or activity of the promoter region of p22(phox) gene are present in SHRs. To carry out this analysis, first of all, we characterized the rat gene structure and promoter region for the p22(phox) subunit. The p22(phox) gene spans approximately 10 kb and contains 6 exons and 5 introns. Primer extension analysis indicated the transcriptional start site 100 bp upstream from the translational start site. The immediate promoter region of the p22(phox) gene does not contain a TATA box, but there are a CCAC box and putative recognition sites for nuclear factors, such as SP1, gamma-interferon, and nuclear factor-kappaB. Using reporter-gene transfection analysis, we found that this promoter was functional in VSMCs. Furthermore, we observed that p22(phox) promoter activity was significantly higher in VSMCs from SHRs than from normotensive Wistar-Kyoto rats. In addition, we found that there were 5 polymorphisms in the sequence of p22(phox) promoter between Wistar-Kyoto rats and SHRs and that they were functional. The results obtained in this study provide a tool to explore the mechanisms that regulate the expression of p22(phox) gene in rat VSMCs. Furthermore, our findings show that changes in the sequence of p22(phox) gene promoter and in the degree of activation of VSMCs are responsible for upregulated expression of p22(phox) in SHRs

Altered cardiac expression of peroxisome proliferator-activated receptor-isoforms in patients with hypertensive heart disease

OBJECTIVE: To investigate whether cardiac expression of the nuclear peroxisome proliferator-activated receptor alpha (PPARalpha) is altered in patients with hypertensive heart disease (HHD). METHODS: We studied endomyocardial septal biopsies from 24 patients with essential hypertension divided into three groups: 6 without left ventricular hypertrophy (LVH) (HT group), 10 with LVH (LVH group), and 8 with LVH and heart failure (HF) (HF group). The expression of two PPARalpha isoforms (the native active and the truncated inhibitory) was analyzed by Western blot and reverse transcription polymerase chain reaction (RT-PCR), and two PPARalpha target genes were evaluated by RT-PCR. Histomorphological features were evaluated in a second myocardial sample from LVH and HF groups. RESULTS: Whereas the expression of native PPARalpha protein was lower (p<0.05) in LVH and HF groups than in the HT group, truncated PPARalpha protein was overexpressed (p<0.001) in the HF group as compared with LVH and HT groups. The mRNA expression of native and truncated PPARalpha was similar in the three groups of hypertensives. In addition, a progressive decrease (p for trend<0.05) in the two PPARalpha target genes mRNA expression was observed among HT, LVH and HF groups. The amount of truncated PPARalpha protein correlates directly with cardiomyocytes apoptosis and inversely with cardiomyocytes density in patients with HHD. In addition, the expression of truncated PPARalpha protein was directly correlated with left ventricular volumes, and inversely with ejection fraction in all hypertensives. CONCLUSIONS: These findings suggest that post-transcriptional regulation of PPARalpha isoforms is altered in patients with HHD, namely in those developing HF. An excess of the truncated inhibitory isoform may be involved in hypertensive left ventricular failure and remodeling

Oxidative Stress in Arterial Hypertension: Role of NAD(P)H Oxidase

Increased vascular reactive oxygen species production, especially superoxide anion, contributes significantly in the functional and structural alterations present in hypertension. An enhanced superoxide production causes a diminished NO bioavailability by an oxidative reaction that inactivates NO. Exaggerated superoxide levels and a low NO bioavailability lead to endothelial dysfunction and hypertrophy of vascular cells. It has been shown that the enzyme NAD(P)H oxidase plays a major role as the most important source of superoxide anion in vascular cells. Several experimental observations have shown an enhanced superoxide generation as a result of the activation of vascular NAD(P)H oxidase in hypertension. Although this enzyme responds to stimuli such as vasoactive factors, growth factors, and cytokines, some recent data suggest the existence of a genetic background modulating the expression of its different components. New polymorphisms have been identified in the promoter of the p22(phox) gene, an essential subunit of NAD(P)H oxidase, influencing the activity of this enzyme. Genetic investigations of these polymorphisms will provide novel markers for determination of genetic susceptibility to oxidative stress in hypertension

Vascular NADH/NADPH oxidase is involved in enhanced superoxide production in spontaneously hypertensive rats

This study was designed to test the hypothesis that stimulation of nicotinamide adenine dinucleotide/nicotinamide adenine dinucleotide phosphate (NADH/NADPH) oxidase is involved in increased vascular superoxide anion (*O(2)(-)) production in spontaneously hypertensive rats (SHR). The study was performed in 16-week-old and 30-week-old normotensive Wistar-Kyoto rats (WKY(16) and WKY(30), respectively) and in 16-week-old and 30-week-old SHR (SHR(16) and SHR(30), respectively). In addition, 16-week-old SHR were treated with oral irbesartan (average dose 20 mg/kg per day) for 14 weeks (SHR(30)-I). Aortic NADH/NADPH oxidase activity was determined by use of chemiluminescence with lucigenin. The expression of p22phox messenger RNA was assessed by competitive reverse transcription-polymerase chain reaction. Vascular responses to acetylcholine were determined by isometric tension studies. Aortic wall structure was studied, determining the media thickness and the cross-sectional area by morphometric analysis. Whereas systolic blood pressure was significantly increased in the 2 groups of hypertensive animals compared with their normotensive controls, no differences were observed in systolic blood pressure between SHR(30) and SHR(16). No other differences in the parameters measured were found between WKY(16) and SHR(16). In SHR(30) compared with WKY(30), we found significantly greater p22phox mRNA level, NADH/NADPH-driven *O(2)(-) production, media thickness, and cross-sectional area and an impaired vasodilation in response to acetylcholine. Treated SHR had similar NADH/NADPH oxidase activity and p22phox expression as the WKY(30) group. The vascular functional and morphological parameters were improved in SHR(30)-I. These findings suggest that an association exists between p22phox gene overexpression and NADH/NADPH overactivity in the aortas of adult SHR. Enhanced NADH/NADPH oxidase-dependent *O(2)(-) production may contribute to endothelial dysfunction and vascular hypertrophy in this genetic model of hypertension

p-SMAD2/3 and DICER promote pre-miR-21 processing during pressure overload-associated myocardial remodeling

AbstractTransforming growth factor-β (TGF-β) induces miR-21 expression which contributes to fibrotic events in the left ventricle (LV) under pressure overload. SMAD effectors of TGF-β signaling interact with DROSHA to promote primary miR-21 processing into precursor miR-21 (pre-miR-21). We hypothesize that p-SMAD-2 and -3 also interact with DICER1 to regulate the processing of pre-miR-21 to mature miR-21 in cardiac fibroblasts under experimental and clinical pressure overload. The subjects of the study were mice undergoing transverse aortic constriction (TAC) and patients with aortic stenosis (AS). In vitro, NIH-3T3 fibroblasts transfected with pre-miR-21 responded to TGF-β1 stimulation by overexpressing miR-21. Overexpression and silencing of SMAD2/3 resulted in higher and lower production of mature miR-21, respectively. DICER1 co-precipitated along with SMAD2/3 and both proteins were up-regulated in the LV from TAC-mice. Pre-miR-21 was isolated bound to the DICER1 maturation complex. Immunofluorescence analysis revealed co-localization of p-SMAD2/3 and DICER1 in NIH-3T3 and mouse cardiac fibroblasts. DICER1-p-SMAD2/3 protein–protein interaction was confirmed by in situ proximity ligation assay. Myocardial up-regulation of DICER1 constituted a response to pressure overload in TAC-mice. DICER mRNA levels correlated directly with those of TGF-β1, SMAD2 and SMAD3. In the LV from AS patients, DICER mRNA was up-regulated and its transcript levels correlated directly with TGF-β1, SMAD2, and SMAD3. Our results support that p-SMAD2/3 interacts with DICER1 to promote pre-miR-21 processing to mature miR-21. This new TGFβ-dependent regulatory mechanism is involved in miR-21 overexpression in cultured fibroblasts, and in the pressure overloaded LV of mice and human patients

Critical Role of Oxygen in Silver-Catalyzed Glaser-Hay Coupling on Ag(100) under Vacuum and in Solution on Ag Particles

The essential role of oxygen in enabling heterogeneously catalyzed Glaser-Hay coupling of phenylacetylene on Ag(100) was elucidated by STM, laboratory and synchrotron photoemission, and DFT calculations. In the absence of coadsorbed oxygen, phenylacetylene formed well-ordered dense overlayers which, with increasing temperature, desorbed without reaction. In striking contrast, even at 120 K, the presence of oxygen led to immediate and complete disruption of the organic layer due to abstraction of acetylenic hydrogen with formation of a disordered mixed layer containing immobile adsorbed phenylacetylide. At higher temperatures phenylacetylide underwent Glaser-Hay coupling to form highly ordered domains of diphenyldiacetylene that eventually desorbed without decomposition, leaving the bare metal surface. DFT calculations showed that, while acetylenic H abstraction was otherwise an endothermic process, oxygen adatoms triggered a reaction-initiating exothermic pathway leading to OH(a) + phenylacetylide, consistent with the experimental observations. Moreover, it was found that, with a solution of phenylacetylene in nonane and in the presence of O, Ag particles catalyzed Glaser-Hay coupling with high selectivity. Rigorous exclusion of oxygen from the reactor strongly suppressed the catalytic reaction. Interestingly, too much oxygen lowers the selectivity toward diphenyldiacetylene. Thus, vacuum studies and theoretical calculations revealed the key role of oxygen in the reaction mechanism, subsequently borne out by catalytic studies with Ag particles that confirmed the presence of oxygen as a necessary and sufficient condition for the coupling reaction to occur. The direct relevance of model studies to a mechanistic understanding of coupling reactions under conditions of practical catalysis was reaffirmed.Support from the European Union FEDER Program and MINECO under projects MAT2013-40852-R and 201560E055 is acknowledged. Computational resources were provided by the Spanish Ministerio de Economía y Competitividad, grant CTQ2015-64669-P, and the EU FEDER Program

Burden and challenges of heart failure in patients with chronic kidney disease. A call to action

Patients with the dual burden of chronic kidney disease (CKD) and chronic congestive heart failure (HF) experience unacceptably high rates of symptom load, hospitalization, and mortality. Currently, concerted efforts to identify, prevent and treat HF in CKD patients are lacking at the institutional level, with emphasis still being placed on individual specialty views on this topic. The authors of this review paper endorse the need for a dedicated cardiorenal interdisciplinary team that includes nephrologists and renal nurses and jointly manages appropriate clinical interventions across the inpatient and outpatient settings. There is a critical need for guidelines and best clinical practice models from major cardiology and nephrology professional societies, as well as for research funding in both specialties to focus on the needs of future therapies for HF in CKD patients. The implementation of crossspecialty educational programs across all levels in cardiology and nephrology will help train future specialists and nurses who have the ability to diagnose, treat, and prevent HF in CKD patients in a precise, clinically effective, and cost-favorable manner.Los pacientes con enfermedad renal crónica (ERC) que desarrollan insuficiencia cardíaca (IC) congestiva crónica presentan cifras inaceptablemente altas de síntomas, hospitalización y mortalidad. Actualmente, se echan en falta iniciativas institucionales dirigidas a identificar, prevenir y tratar la IC en los pacientes con ERC de manera multidisciplinar, prevaleciendo las actuaciones de las especialidades individuales. Los autores de este artículo de revisión respaldan la necesidad de crear equipos multidisciplinares cardiorrenales, en los que participen nefrólogos y enfermeras renales, que gestionen colaborativamente las intervenciones clínicas apropiadas en los entornos de pacientes con ERC e IC hospitalizados y ambulatorios. Es necesario y urgente que se elaboren guías y modelos de práctica clínica sobre la ERC con IC por parte de las sociedades profesionales de cardiología y nefrología, así como financiación para la investigación concertada entre ambas especialidades sobre la necesidad de futuros tratamientos para la IC en pacientes con ERC. La implementación de programas educativos cardiorrenales a todos los niveles en cardiología y nefrología ayudará a formar a los futuros especialistas y enfermeras para que tengan la capacidad de diagnosticar, tratar y prevenir la IC en pacientes con ERC de manera precisa, clínicamente efectiva y económicamente favorabl

Lack of replication of interactions between polymorphisms in rheumatoid arthritis susceptibility: case–control study

[Abstract] INTRODUCTION: Approximately 100 loci have been definitively associated with rheumatoid arthritis (RA) susceptibility. However, they explain only a fraction of RA heritability. Interactions between polymorphisms could explain part of the remaining heritability. Multiple interactions have been reported, but only the shared epitope (SE) × protein tyrosine phosphatase nonreceptor type 22 (PTPN22) interaction has been replicated convincingly. Two recent studies deserve attention because of their quality, including their replication in a second sample collection. In one of them, researchers identified interactions between PTPN22 and seven single-nucleotide polymorphisms (SNPs). The other showed interactions between the SE and the null genotype of glutathione S-transferase Mu 1 (GSTM1) in the anti-cyclic citrullinated peptide-positive (anti-CCP+) patients. In the present study, we aimed to replicate association with RA susceptibility of interactions described in these two high-quality studies. METHODS: A total of 1,744 patients with RA and 1,650 healthy controls of Spanish ancestry were studied. Polymorphisms were genotyped by single-base extension. SE genotypes of 736 patients were available from previous studies. Interaction analysis was done using multiple methods, including those originally reported and the most powerful methods described. RESULTS: Genotypes of one of the SNPs (rs4695888) failed quality control tests. The call rate for the other eight polymorphisms was 99.9%. The frequencies of the polymorphisms were similar in RA patients and controls, except for PTPN22 SNP. None of the interactions between PTPN22 SNPs and the six SNPs that met quality control tests was replicated as a significant interaction term--the originally reported finding--or with any of the other methods. Nor was the interaction between GSTM1 and the SE replicated as a departure from additivity in anti-CCP+ patients or with any of the other methods. CONCLUSIONS: None of the interactions tested were replicated in spite of sufficient power and assessment with different assays. These negative results indicate that whether interactions are significant contributors to RA susceptibility remains unknown and that strict standards need to be applied to claim that an interaction exists.Instituto de Salud Carlos III; 11/01048Instituto de Salud Carlos III; 12/01909Instituto de Salud Carlos III; RD12/0009/000

Lack of replication of interactions between polymorphisms in rheumatoid arthritis susceptibility: case-control study

Introduction: Approximately 100 loci have been definitively associated with rheumatoid arthritis (RA) susceptibility. However, they explain only a fraction of RA heritability. Interactions between polymorphisms could explain part of the remaining heritability. Multiple interactions have been reported, but only the shared epitope (SE) × protein tyrosine phosphatase nonreceptor type 22 (PTPN22) interaction has been replicated convincingly. Two recent studies deserve attention because of their quality, including their replication in a second sample collection. In one of them, researchers identified interactions between PTPN22 and seven single-nucleotide polymorphisms (SNPs). The other showed interactions between the SE and the null genotype of glutathione S-transferase Mu 1 (GSTM1) in the anti-cyclic citrullinated peptide-positive (anti-CCP+) patients. In the present study, we aimed to replicate association with RA susceptibility of interactions described in these two high-quality studies. Methods: A total of 1,744 patients with RA and 1,650 healthy controls of Spanish ancestry were studied. Polymorphisms were genotyped by single-base extension. SE genotypes of 736 patients were available from previous studies. Interaction analysis was done using multiple methods, including those originally reported and the most powerful methods described. Results: Genotypes of one of the SNPs (rs4695888) failed quality control tests. The call rate for the other eight polymorphisms was 99.9%. The frequencies of the polymorphisms were similar in RA patients and controls, except for PTPN22 SNP. None of the interactions between PTPN22 SNPs and the six SNPs that met quality control tests was replicated as a significant interaction term the originally reported finding or with any of the other methods. Nor was the interaction between GSTM1 and the SE replicated as a departure from additivity in anti-CCP+ patients or with any of the other methods. Conclusions: None of the interactions tested were replicated in spite of sufficient power and assessment with different assays. These negative results indicate that whether interactions are significant contributors to RA susceptibility remains unknown and that strict standards need to be applied to claim that an interaction exists