Inter-rhombomeric interactions reveal roles for fibroblast growth factors signaling in segmental regulation of EphA4 expression.

BACKGROUND: The basic ground plan of vertebrate hindbrain is established through a process of segmentation, which generates eight transient lineage-restricted cellular compartments called rhombomeres (r). The segments adopt distinct individual identities in response to axial patterning signals. It is unclear whether signaling between rhombomeres plays a conserved role in regulating segmental patterning during hindbrain development. RESULTS: Using tissue manipulations of rhombomeres in chicken embryos, we have uncovered roles for r2 and r4 in regulating the expression of EphA4 in r3 and r5. Perturbations of signaling pathways reveal that these regulatory inputs from r2 and r4 into EphA4 expression are mediated independent of inputs from Krox20 through cues involving fibroblast growth factor (FGF) signaling. These interactions are stage dependent and are set up in embryos with <10 somites. CONCLUSIONS: We show that r2 and r4 function as temporally dynamic signaling centers in the early patterning of adjacent hindbrain segments and this activity is dependent upon the FGF pathway. These results reveal that inter-rhombomeric signaling is a conserved feature of the regulatory networks that control the specification of individual rhombomere identities in vertebrate hindbrain segmentation. However, the timing of when restricted domains of FGF signaling are coupled to formation of r4 may vary between the species.

Prevalencia y determinantes clínicos de ecocardiografía de esfuerzo no concluyente en pacientes con dolor torácico / Inconclusive exercise stress echocardiography in patients with chest pain: prevalence and clinical determinants

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Mild kidney disease as a risk factor for major bleeding in patients with atrial fibrillation undergoing percutaneous coronary stenting.

Bleeding risk is increased in patients with atrial fibrillation (AF) and moderate to severe kidney disease (KD); however, the implication of mild KD on bleeding remains unclear. The aim of this study was to determine whether the presence of mild KD increases risk for major bleeding (MB) in patients with AF undergoing percutaneous coronary intervention with stent implantation (PCI-S). Two hundred eighty-five patients were included. Patients were classified into three kidney function groups: moderate to severe KD (n=91; <60 ml/min/1.73 m²), mild KD (n=139; 60-89 ml/min/1.73 m²) and non-KD (n=55; ≥90 ml/min/1.73 m²). Estimated glomerular filtration rate was calculated using the simplified Modification of Diet in Renal Disease equation. Patients were followed for one year, and the occurrence of MB was obtained in all. A total of 28 patients (9.8%) presented MB. MB complications examined as a function of KD groups revealed that there was a graded increase in MB with worsening renal function (non KD=1.8%, mild KD=7.9%, moderate to severe KD=17.6%; p <0.001). Multivariable Cox regression analysis showed that mild KD was associated with nearly a 2.5-fold (2.43 95% confidence interval 1.11-5.34, p=0.039) increase in the risk of MB as compared with non-KD patients. Other independent predictors of MB were moderate-severe KD, anaemia and triple antithrombotic therapy after PCI-S (C-index=0.76). In this population, mild KD confers a significantly increase in the risk for MB complications. Future studies should assess the potential role of incorporating mild KD into the bleeding risk scales to improve the stratification of these patients.

Serum levels of high-sensitivity troponin T: a novel marker for cardiac remodeling in hypertrophic cardiomyopathy.

BACKGROUND: Hypertrophic cardiomyopathy (HCM) is characterized by inappropriate hypertrophy, small-vessel coronary artery disease, myocyte disarray, and increased interstitial fibrosis. High-sensitivity troponin T (hs-TnT) could be a reliable indicator of myocardial remodeling, a proposed prognostic marker in HCM. Therefore we hypothesized that increased hs-TnT levels are related to different variables associated with myocardial remodeling, such as the presence of fibrosis assessed with cardiac magnetic resonance imaging (MRI). METHODS AND RESULTS: We included 95 hemodynamically stable HCM patients, 72 male, aged 45.7 ± 14.2 years, and 45 healthy control subjects with similar age and gender. A complete history and clinical examination was performed, including 12-lead electrocardiogram (ECG), echocardiography, 24-hour ECG-Holter monitoring, symptom-limited treadmill exercise test, and late gadolinium enhancement in cardiac MRI. Risk factors for sudden death were evaluated. A blinded cardiac MRI was performed with late gadolinium enhancement study. Serum hs-TnT levels were assayed. A high proportion (42%) of hemodynamically stable patients studied showed increased levels of hs-TnT. The hs-TnT levels were raised in patients with severe dyspnea: New York Heart Association (NYHA) functional class ≥3 (P = .020), outflow obstruction (P = .013), systolic dysfunction (P = .037), abnormal blood pressure response (P = .036), and presence of gadolinium enhancement (P = .021). The hs-TnT levels correlated positively with the maximum left ventricular wall thickness (r = 0.47; P < .001), left atrial diameter (r = 0.36, P = .014), and outflow gradient (r = 0.28; P = .008). CONCLUSIONS: A high proportion of hemodynamically stable patients show increased levels of hs-TnT. We observed that raised hs-TnT serum levels are associated with different conditions related to the severity of the disease.

Plasma levels of von Willebrand factor are increased in patients with hypertrophic cardiomyopathy.

UNLABELLED: Hypertrophic cardiomyopathy (HCM) is characterised by inappropriate hypertrophy, small-vessel coronary artery disease, myocyte disarray and increased interstitial fibrosis. Microvascular dysfunction is a common finding in HCM and its extent has been proposed as an important prognostic marker. Plasma von Willebrand factor (vWf) is an established marker of endothelial damage or dysfunction; however it has scarcely been studied in HCM. We hypothesised that vWf could be raised in patients with HCM and be related to different variables associated with severity of HCM. METHODS: We included 124 HCM patients, 93 males, aged 48+/-15 years, 59 healthy control subjects with similar age and sex and 20 patients with ischemic heart disease but clinical stability for the last 6 months. A complete history and clinical examination was performed, including 12-lead electrocardiogram, echocardiography, 24 hours ECG-Holter monitoring, and symptom limited treadmill exercise test. Risk factors for sudden death were evaluated. A blinded cardiac MRI was performed with late enhanced study with Gadolinium. Plasma vWf levels were assayed by commercial ELISA. RESULTS: Patients showed higher levels of vWf (140.0+/-65.0 UI/ml vs 105.0+/-51.0 UI/ml, p<0.001) even after adjusting for ABO blood group. vWf levels were found raised in patients with severe functional class (168.4+/-65.9 UI/mL vs 132.4+/-60.7 UI/mL, p=0.020), atrial fibrillation (175.8+/-69.4 UI/mL vs 133.0+/-59.0 UI/mL, p=0.005), hypertension (161.4+/-60.8 vs 128.9+/-60.5, p=0.010) obstruction (153.9+/-67.9 vs 128.2+/-57.4 UI/mL, p=0.046) and non sustained ventricular tachycardia (159.3+/-59.1 vs 133.0+/-63.0, p=0.049). vWf correlated with age (r:0.26; p=0.006) and obstruction (r:0.22; p=0.021). CONCLUSIONS: We show, for the first time, patients with HCM present significantly raised levels of vWf. These are associated with different conditions related to the severity of the disease.

An atypical presentation of Tako-Tsubo cardiomyopathy.

We present the case of a patient with Tako-Tsubo cardiomyopathy whose initial diagnosis, based on the location of shoulder and chest pain and electrocardiographic (ECG) changes, suggested that she was suffering from pericarditis. However, 24 h after admission, evolutionary changes of ECG and the echocardiogram performed suggested a Tako-Tsubo cardiomyopathy. In this context, we review the literature to discuss the clinical presentation and evolutionary ECG changes associated with Tako-Tsubo cardiomyopathy.

Does pacemaker implantation provide long-term benefits in severe obstructive hypertrophic cardiomyopathy?

INTRODUCTION AND OBJECTIVES: About 25% of patients with obstructive hypertrophic cardiomyopathy (HCM) remain symptomatic despite optimal medical treatment. Some may benefit from pacemaker implantation. The aim of this study was to determine the effect of pacemaker implantation on the left ventricular outflow tract (LVOT) gradient, the maximum thickness of the left ventricle, and functional capacity. METHODS: In total, 72 patients with obstructive HCM and incapacitating symptoms underwent pacemaker implantation. Clinical examination, echocardiography (in 61 patients) and treadmill testing (in 34 patients) were performed before and after implantation. RESULTS: Subjective functional capacity, as assessed using the New York Heart Association (NYHA) classification, improved in 43.1% of patients, but treadmill testing showed no change. There were significant reductions in subaortic gradient, from a median of 87.0 mmHg (interquartile range [IQR] 61.5-115.2 mmHg) to 30.0 mmHg (IQR 18.0-54.5 mmHg; P< .001), and maximum left ventricular thickness, from 22.1+/-4.5 mm to 19.8+/-3.6 mm (P=.001). Univariate analysis identified two factors associated with clinical improvement: female sex (odds ratio [OR]=3.43; P=.020) and functional class III/IV (OR=4.17; P=.009). On multivariate analysis, only functional class III/IV remained a significant predictor (OR=3.12; P=.048). CONCLUSIONS: In patients with obstructive HCM and incapacitating symptoms, pacemaker implantation reduced the LVOT gradient and the maximum left ventricular thickness, but only 43.1% of patients experienced clinical improvement. The only factor predictive of improvement was advanced NYHA functional class.

¿Existe un efecto beneficioso a largo plazo con el tratamiento con marcapasos de la miocardiopatía hipertrófica obstructiva severa? / Does pacemaker implantation provide long-term benefits in severe obstructive hypertrophic cardiomyopathy?

Introducción y objetivos. Alrededor de un 25% de los pacientes con MCH obstructiva permanecen sintomáticos a pesar de una correcta medicación. Algunos pueden beneficiarse del implante de un marcapasos. El objetivo fue valorar el efecto del marcapasos en la modificación del gradiente en el tracto de salida del ventrículo izquierdo (TSVI), grosor máximo del ventrículo izquierdo (VI) y en la capacidad funcional. Métodos. A 72 pacientes con MCH obstructiva y síntomas incapacitantes se les implantó un marcapasos. Se realizó un examen clínico, una ecocardiografía (61 pacientes) y una ergometría (34 pacientes) antes y después de la implantación del marcapasos. Resultados. La capacidad funcional subjetiva, estimada según la clasificación de la NYHA, mejoró en el 43,1% de los pacientes, aunque no lo hizo la estimada mediante ergometría. Se observó una reducción significativa del gradiente subaórtico (mediana, 87 [intervalo intercuartílico, 61,5-115,2] frente a 30 [18-54,5] mmHg; p < 0,001) y del grosor máximo del VI (22,1 ± 4,5 frente a 19,8 ± 3,6 mm; p = 0,001). En el análisis univariable, el sexo femenino (OR = 3,43; p = 0,020) y la clase funcional III/IV (OR = 4,17; p = 0,009) se asociaron a una mejoría clínica. En el análisis multivariable, sólo la clase funcional III/IV mantuvo la significación (OR = 3,12; p = 0,048). Conclusiones. La implantación de marcapasos en pacientes con MCH obstructiva con síntomas incapacitantes disminuye el gradiente obstructivo del TSVI y el grosor máximo del VI, pero sólo el 43,1% consigue una mejoría clínica subjetiva, siendo una clase funcional más avanzada el único factor predictor de mejoría (AU)

Introduction and objectives. About 25% of patients with obstructive hypertrophic cardiomyopathy (HCM) remain symptomatic despite optimal medical treatment. Some may benefit from pacemaker implantation. The aim of this study was to determine the effect of pacemaker implantation on the left ventricular outflow tract (LVOT) gradient, the maximum thickness of the left ventricle, and functional capacity. Methods. In total, 72 patients with obstructive HCM and incapacitating symptoms underwent pacemaker implantation. Clinical examination, echocardiography (in 61 patients) and treadmill testing (in 34 patients) were performed before and after implantation. Results. Subjective functional capacity, as assessed using the New York Heart Association (NYHA) classification, improved in 43.1% of patients, but treadmill testing showed no change. There were significant reductions in subaortic gradient, from a median of 87.0 mmHg (interquartile range [IQR] 61.5-115.2 mmHg) to 30.0 mmHg (IQR 18.0-54.5 mmHg; P < .001), and maximum left ventricular thickness, from 22.1±4.5 mm to 19.8±3.6 mm (P=.001). Univariate analysis identified two factors associated with clinical improvement: female sex (odds ratio [OR]=3.43; P=.020) and functional class III/IV (OR=4.17; P=.009). On multivariate analysis, only functional class III/IV remained a significant predictor (OR=3.12; P=.048). Conclusions. In patients with obstructive HCM and incapacitating symptoms, pacemaker implantation reduced the LVOT gradient and the maximum left ventricular thickness, but only 43.1% of patients experienced clinical improvement. The only factor predictive of improvement was advanced NYHA functional class (AU)

Biomarkers of pathophysiology in hypertrophic cardiomyopathy: implications for clinical management and prognosis.

The study of biomarkers and their signalling pathways has allowed the development of new therapeutic strategies in a range of disorders. The aim of the present systematic review is to provide an overview of different biomarkers in patients with hypertrophic cardiomyopathy that could give some insight into the pathophysiologic mechanism(s) underlying the typical clinical and histological manifestations of the disease. Several pathophysiological models are presented and discussed, including studies that have investigated these biomarkers for diagnostic and prognostic reasons, in relation to disease progression and/or mortality.

Impact of chronic kidney disease on major bleeding complications and mortality in patients with indication for oral anticoagulation undergoing coronary stenting.

BACKGROUND: Patients with indications for oral anticoagulation (OAC) undergoing percutaneous coronary artery stenting (PCI-S) represent a high-risk population for major bleeding complications. Chronic kidney disease (CKD) is also associated with poor outcome after PCI-S. Limited data are available regarding the impact of CKD on the frequency of major bleeding and mortality in this population. METHODS: We investigated the influence of CKD on major bleeding and all-cause mortality in patients with indication for OAC who undergo PCI-S. Patients were grouped according to calculated creatinine clearance (CrCl): CrCl > 60 mL/min, (n = 98) and CrCl < or = 60 mL/min, (n = 68). Major bleeding and major adverse vascular events (all-cause mortality, myocardial infarction, repeat revascularization, stent thrombosis, or stroke) were collected during follow-up. RESULTS: We analyzed 166 consecutive patients with indication(s) for OAC (77% men; mean age, 71 years; range, 66 to 76 years) after undergoing PCI-S. CKD was associated with higher risk for major bleeding (hazard ratio [HR], 3.44; 95% confidence interval [CI], 1.50 to 7.93; p = 0.004) and all-cause mortality (HR, 3.50; 95% CI, 1.53 to 7.99; p = 0.003). In multivariate analyses, age > 75 years (HR, 2.75; 95% CI, 1.15 to 6.56; p = 0.023), CKD (HR, 2.59; 95% CI, 1.00 to 6.95; p = 0.049), anemia (HR, 2.36; 95% CI, 1.00 to 5.54; p = 0.049), and triple antithrombotic therapy (HR, 3.29; 95% CI, 1.23 to 8.84; p = 0.018) were independent predictors for major bleeding, whereas age > 75 years (HR, 2.38; 95% CI, 1.03 to 5.59; p = 0.046) and CKD (HR, 2.44; 95% CI, 1.03 to 5.82; p = 0.044) were predictors for all-cause mortality. CONCLUSION: In this high-risk population, CKD is independently associated with increased major bleeding and all-cause mortality following PCI-S.

A regulatory module embedded in the coding region of Hoxa2 controls expression in rhombomere 2.

Here, we define a gene regulatory network for Hoxa2, responsible for temporal and spatial expression in hindbrain development. Hoxa2 plays an important role in regulating the regional identity of rhombomere 2 (r2) and is the only Hox gene expressed in this segment. In this study, we found that a Hoxa2 cis-regulatory module consists of five elements that direct expression in r2 of the developing hindbrain. Surprisingly, the module is imbedded in the second coding exon of Hoxa2 and therefore may be constrained by both protein coding and gene regulatory requirements. This highly conserved enhancer consists of two consensus Sox binding sites and several additional elements that act in concert to direct strong r2 specific expression. Our findings provide important insight into the regulation of segmental identity in the anterior hindbrain. Furthermore, they have broader implications in designing arrays and interpreting data from global analyses of gene regulation because regulatory input from coding regions needs to be considered.

Increased major bleeding complications related to triple antithrombotic therapy usage in patients with atrial fibrillation undergoing percutaneous coronary artery stenting.

BACKGROUND: The optimal antithrombotic therapy strategy for atrial fibrillation (AF) patients who undergo percutaneous coronary intervention with stent implantation (PCI-S) is unknown. We assessed the safety of antithrombotic therapy strategies in AF patients with indication for oral anticoagulation (OAC) undergoing PCI-S. METHODS: We studied consecutive AF patients with indication for OAC who underwent PCI-S. We compared patients that received triple antithrombotic therapy (TT) [aspirin, clopidogrel, and coumadin] against other regimes (non-TT) after PCI-S. The primary end point was defined as the occurrence of major bleeding complications that were termed as early major bleeding (EMB) [< or = 48 h] or late major bleeding (LMB) [> 48 h]. Clinical follow-up was performed, and complications were recorded. RESULTS: We studied 104 patients (mean age +/- SD, 72 +/- 8 years; 70% men); TT was used in 51 patients (49%). TT was associated with a higher incidence of LMB (21.6% vs non-TT, 3.8%; p = 0.006) but not of EMB (5.8% vs non-TT, 11.3%; p = 0.33). In multivariate analyses, glycoprotein (GP) IIb/IIIa inhibitor use (hazard ratio [HR], 13.5; 95% confidence interval [CI], 1.7 to 108.3; p = 0.014) and PCI-S of three vessels or left main artery disease (HR, 7.9; 95% CI, 1.6 to 39.2; p = 0.01) were independent predictors for EMB. TT use (HR, 7.1; 95% CI, 1.5 to 32.4; p = 0.012), the occurrence of EMB (HR, 6.7; 95% CI, 1.8 to 25.3; p = 0.005), and baseline anemia (HR, 3.8; 95% CI, 1.2 to 12.5; p = 0.027) were independent predictors for LMB. No differences in major cardiovascular events were observed in patients treated with TT vs non-TT (25.5% vs 21.0%; p = 0.53). CONCLUSION: A high rate of major bleeding is observed in AF patients with indication for OAC undergoing PCI-S who receive TT. GP IIb/IIIa inhibitor use and multivessel/left main artery disease during PCI-S were independent predictors for EMB, while TT use, occurrence of EMB, and baseline anemia were independent predictors for LMB.

Expression of Hoxa2 in rhombomere 4 is regulated by a conserved cross-regulatory mechanism dependent upon Hoxb1.

The Hoxa2 gene is an important component of regulatory events during hindbrain segmentation and head development in vertebrates. In this study we have used sequenced comparisons of the Hoxa2 locus from 12 vertebrate species in combination with detailed regulatory analyses in mouse and chicken embryos to characterize the mechanistic basis for the regulation of Hoxa2 in rhombomere (r) 4. A highly conserved region in the Hoxa2 intron functions as an r4 enhancer. In vitro binding studies demonstrate that within the conserved region three bipartite Hox/Pbx binding sites (PH1-PH3) in combination with a single binding site for Pbx-Prep/Meis (PM) heterodimers co-operate to regulate enhancer activity in r4. Mutational analysis reveals that these sites are required for activity of the enhancer, suggesting that the r4 enhancer from Hoxa2 functions in vivo as a Hox-response module in combination with the Hox cofactors, Pbx and Prep/Meis. Furthermore, this r4 enhancer is capable of mediating a response to ectopic HOXB1 expression in the hindbrain. These findings reveal that Hoxa2 is a target gene of Hoxb1 and permit us to develop a gene regulatory network for r4, whereby Hoxa2, along with Hoxb1, Hoxb2 and Hoxa1, is integrated into a series of auto- and cross-regulatory loops between Hox genes. These data highlight the important role played by direct cross-talk between Hox genes in regulating hindbrain patterning.

Evolution of cis elements in the differential expression of two Hoxa2 coparalogous genes in pufferfish (Takifugu rubripes).

Sequence divergence in cis-regulatory elements is an important mechanism contributing to functional diversity of genes during evolution. Gene duplication and divergence provide an opportunity for selectively preserving initial functions and evolving new activities. Many vertebrates have 39 Hox genes organized into four clusters (Hoxa-Hoxd); however, some ray-finned fishes have extra Hox clusters. There is a single Hoxa2 gene in most vertebrates, whereas fugu (Takifugu rubripes) and medaka (Oryzias latipes) have two coparalogous genes [Hoxa2(a) and Hoxa2(b)]. In the hindbrain, both genes are expressed in rhombomere (r) 2, but only Hoxa2(b) is expressed in r3, r4, and r5. Multiple regulatory modules directing segmental expression of chicken and mouse Hoxa2 genes have been identified, and each module is composed of a series of discrete elements. We used these modules to investigate the basis of differential expression of duplicated Hoxa2 genes, as a model for understanding the divergence of cis-regulatory elements. Therefore, we cloned putative regulatory regions of the fugu and medaka Hoxa2(a) and -(b) genes and assayed their activity. We found that these modules direct reporter expression in a chicken assay, in a manner corresponding to their endogenous expression pattern in fugu. Although sequence comparisons reveal many differences between the two coparalogous genes, specific subtle changes in seven cis elements of the Hoxa2(a) gene restore segmental regulatory activity. Therefore, drift in subsets of the elements in the regulatory modules is responsible for the differential expression of the two coparalogous genes, thus providing insight into the evolution of cis elements.

Hoxb1 enhancer and control of rhombomere 4 expression: complex interplay between PREP1-PBX1-HOXB1 binding sites.

The Hoxb1 autoregulatory enhancer directs segmental expression in vertebrate hindbrain. Three conserved repeats (R1, R2, and R3) in the enhancer have been described as Pbx-Hoxb1 (PH) binding sites, and one Pbx-Meinox (PM) binding site has also been characterized. We have investigated the importance and relative roles of PH and PM binding sites with respect to protein interactions and in vivo regulatory activity. We have identified a new PM site (PM2) and found that it cooperates with the R3 PH site to form ternary Prep1-Pbx1-Hoxb1 complexes. In vivo, the combination of the R3 and PM2 sites is sufficient to mediate transgenic reporter activity in the developing chick hindbrain. In both chicken and mouse transgenic embryos, mutations of the PM1 and PM2 sites reveal that they cooperate to modulate in vivo regulatory activity of the Hoxb1 enhancer. Furthermore, we have shown that the R2 motif functions as a strong PM site, with a high binding affinity for Prep1-Pbx1 dimers, and renamed this site R2/PM3. In vitro R2/PM3, when combined with the PM1 and R3 motifs, inhibits ternary complex formation mediated by these elements and in vivo reduces and restricts reporter expression in transgenic embryos. These inhibitory effects appear to be a consequence of the high PM binding activity of the R2/PM3 site. Taken together, our results demonstrate that the activity of the Hoxb1 autoregulatory enhancer depends upon multiple Prep1-Pbx1 (PM1, PM2, and PM3) and Pbx1-Hoxb1 (R1 and R3) binding sites that cooperate to modulate and spatially restrict the expression of Hoxb1 in r4 rhombomere.