Peter A. Kavsak

Smad7 Binds to Smurf2 to Form an E3 Ubiquitin Ligase that Targets the TGFβ Receptor for Degradation

Ubiquitin-mediated proteolysis regulates the activity of diverse receptor systems. Here, we identify Smurf2, a C2-WW-HECT domain ubiquitin ligase and show that Smurf2 associates constitutively with Smad7. Smurf2 is nuclear, but binding to Smad7 induces export and recruitment to the activated TGF beta receptor, where it causes degradation of receptors and Smad7 via proteasomal and lysosomal pathways. IFN gamma, which stimulates expression of Smad7, induces Smad7-Smurf2 complex formation and increases TGF beta receptor turnover, which is stabilized by blocking Smad7 or Smurf2 expression. Furthermore, Smad7 mutants that interfere with recruitment of Smurf2 to the receptors are compromised in their inhibitory activity. These studies thus define Smad7 as an adaptor in an E3 ubiquitin-ligase complex that targets the TGF beta receptor for degradation.

A SMAD ubiquitin ligase targets the BMP pathway and affects embryonic pattern formation

The TGF-β superfamily of proteins regulates many different biological processes, including cell growth, differentiation and embryonic pattern formation. TGF-β-like factors signal across cell membranes through complexes of transmembrane receptors known as type I and type II serine/threonine-kinase receptors, which in turn activate the SMAD signalling pathway,. On the inside of the cell membrane, a receptor-regulated class of SMADs are phosphorylated by the type-I-receptor kinase. In this way, receptors for different factors are able to pass on specific signals along the pathway: for example, receptors for bone morphogenetic protein (BMP) target SMADs 1, 5 and 8, whereas receptors for activin and TGF-β target SMADs 2 and 3. Phosphorylation of receptor-regulated SMADs induces their association with Smad4, the ‘common-partner’ SMAD, and stimulates accumulation of this complex in the nucleus, where it regulates transcriptional responses. Here we describe Smurf1, a new member of the Hect family of E3 ubiquitin ligases. Smurf1 selectively interacts with receptor-regulated SMADs specific for the BMP pathway in order to trigger their ubiquitination and degradation, and hence their inactivation. In the amphibian Xenopus laevis, Smurf1 messenger RNA is localized to the animal pole of the egg; in Xenopus embryos, ectopic Smurf1 inhibits the transmission of BMP signals and thereby affects pattern formation. Smurf1 also enhances cellular responsiveness to the Smad2 (activin/TGF-β) pathway. Thus, targeted ubiquitination of SMADs may serve to control both embryonic development and a wide variety of cellular responses to TGF-β signals.

TGF-β induces assembly of a Smad2- Smurf2 ubiquitin ligase complex that targets SnoN for degradation

The receptor-regulated Smad proteins are essential intracellular mediators of signal transduction by the transforming growth factor-β (TGF-β) superfamily of growth factors and are also important as regulators of gene transcription. Here we describe a new role for TGF-β-regulated Smad2 and Smad3 as components of a ubiquitin ligase complex. We show that in the presence of TGF-β signalling, Smad2 interacts through its proline-rich PPXY motif with the tryptophan-rich WW domains of Smurf2, a recently identified E3 ubiquitin ligases. TGF-β also induces the association of Smurf2 with the transcriptional co-repressor SnoN and we show that Smad2 can function to mediate this interaction. This allows Smurf2 HECT domain to target SnoN for ubiquitin-mediated degradation by the proteasome. Thus, stimulation by TGF-β can induce the assembly of a Smad2–Smurf2 ubiquitin ligase complex that functions to target substrates for degradation.

Short- and Long-Term Risk Stratification Using a Next-Generation, High-Sensitivity Research Cardiac Troponin I (hs-cTnI) Assay in an Emergency Department Chest Pain Population

BACKGROUND The next-generation, high-sensitivity cardiac troponin assays can measure quantifiable concentrations of cTn in a majority of individuals, but there are few studies assessing these assays for risk stratification. The present study was undertaken to determine if a research hs-cTnI assay can be useful for predicting death/myocardial infarction (MI), both short- and long-term, in an emergency department acute coronary syndrome (ACS) population. METHODS In a cohort of 383 subjects, originally recruited in 1996, presenting to the emergency department with symptoms suggestive of ACS, the heparin plasma obtained at initial presentation was thawed and measured in 2007 with a research hs-cTnI assay. AccuTnI (Beckman Coulter) measurements were made on these same samples in 2003. The population was divided into 4 groups by hs-cTnI: <5.00, 5.00-9.99, 10.00-40.00, and >40.00 ng/L. Kaplan-Meier, Cox proportional hazards, ROC curves, and logistic regression analyses were used to identify which hs-cTnI concentrations were predictive of death/MI within 10 years after presentation. RESULTS There were significant differences between the hs-cTnI groups for the probability of death/MI up to 10 years after presentation (P < 0.05). At 6 months, patients with hs-cTnI > or =10.00 ng/L were at higher risk for death/MI (hazard ratio >3.7; P < 0.05) compared with those having hs-cTnI <5.00 ng/L. ROC curve analysis for death/MI at 30 days with the hs-cTnI assay had an area under the curve of 0.74 (95% CI 0.65-0.82), with logistic models yielding an optimal assay threshold of 12.68 ng/L. CONCLUSIONS This research hs-cTnI assay appears useful for risk stratification for death/MI in an ACS population.

High-Sensitivity Cardiac Troponin I Measurement for Risk Stratification in a Stable High-Risk Population

BACKGROUND Past investigations regarding the utility of high-sensitivity cardiac troponin I (cTnI) assays have been focused primarily on the acute coronary syndrome setting. We assessed whether such assays can predict future ischemic cardiovascular events in a stable high-risk population. METHODS We quantified serum cTnI using an investigational high-sensitivity assay (hs-cTnI IUO, Beckman Coulter) in 2572 participants from the Heart Outcomes Prevention Evaluation (HOPE) study. The derived ROC curve cutoff and the 99th percentile for the hs-cTnI assay were assessed by Kaplan-Meier and Cox analyses for the primary outcome [composite of myocardial infarction (MI), stroke, and cardiovascular death] at 4.5 years of follow-up. We also assessed individual outcomes (MI, stroke, cardiovascular death) and the combined outcome (MI/cardiovascular death) by regression analyses to determine hazard ratios (HRs) and c statistics in models that included established risk factors, C-reactive protein, and N-terminal pro-B-type natriuretic peptide (NT-proBNP). RESULTS Participants with hs-cTnI >6 ng/L (ROC cutoff) were at higher risk for the primary outcome (HR 1.38, 95% CI 1.09-1.76; P = 0.008, adjusted models). For the individual outcomes, participants with hs-cTnI above the 99th percentile (≥10 ng/L) had higher risk for cardiovascular death (HR 2.15, 95% CI 1.32-3.52; P = 0.002) and MI (HR 1.49, 95% CI 1.05-2.10; P = 0.025) but not stroke (HR 1.38, 95% CI 0.76-2.47; P = 0.288, adjusted models). Addition of hs-cTnI to an established risk model with NT-proBNP also yielded a higher c statistic for the combined outcome of MI/cardiovascular death. CONCLUSIONS The investigational Beckman Coulter hs-cTnI assay provides prognostic information for future MI and cardiovascular death in a stable high-risk population.

Association of Postoperative High-Sensitivity Troponin Levels With Myocardial Injury and 30-Day Mortality Among Patients Undergoing Noncardiac Surgery

Importance Little is known about the relationship between perioperative high-sensitivity troponin T (hsTnT) measurements and 30-day mortality and myocardial injury after noncardiac surgery (MINS). Objective To determine the association between perioperative hsTnT measurements and 30-day mortality and potential diagnostic criteria for MINS (ie, myocardial injury due to ischemia associated with 30-day mortality). Design, Setting, and Participants Prospective cohort study of patients aged 45 years or older who underwent inpatient noncardiac surgery and had a postoperative hsTnT measurement. Starting in October 2008, participants were recruited at 23 centers in 13 countries; follow-up finished in December 2013. Exposures Patients had hsTnT measurements 6 to 12 hours after surgery and daily for 3 days; 40.4% had a preoperative hsTnT measurement. Main Outcomes and Measures A modified Mazumdar approach (an iterative process) was used to determine if there were hsTnT thresholds associated with risk of death and had an adjusted hazard ratio (HR) of 3.0 or higher and a risk of 30-day mortality of 3% or higher. To determine potential diagnostic criteria for MINS, regression analyses ascertained if postoperative hsTnT elevations required an ischemic feature (eg, ischemic symptom or electrocardiography finding) to be associated with 30-day mortality. Results Among 21 842 participants, the mean age was 63.1 (SD, 10.7) years and 49.1% were female. Death within 30 days after surgery occurred in 266 patients (1.2%; 95% CI, 1.1%-1.4%). Multivariable analysis demonstrated that compared with the reference group (peak hsTnT <5 ng/L), peak postoperative hsTnT levels of 20 to less than 65 ng/L, 65 to less than 1000 ng/L, and 1000 ng/L or higher had 30-day mortality rates of 3.0% (123/4049; 95% CI, 2.6%-3.6%), 9.1% (102/1118; 95% CI, 7.6%-11.0%), and 29.6% (16/54; 95% CI, 19.1%-42.8%), with corresponding adjusted HRs of 23.63 (95% CI, 10.32-54.09), 70.34 (95% CI, 30.60-161.71), and 227.01 (95% CI, 87.35-589.92), respectively. An absolute hsTnT change of 5 ng/L or higher was associated with an increased risk of 30-day mortality (adjusted HR, 4.69; 95% CI, 3.52-6.25). An elevated postoperative hsTnT (ie, 20 to <65 ng/L with an absolute change ≥5 ng/L or hsTnT ≥65 ng/L) without an ischemic feature was associated with 30-day mortality (adjusted HR, 3.20; 95% CI, 2.37-4.32). Among the 3904 patients (17.9%; 95% CI, 17.4%-18.4%) with MINS, 3633 (93.1%; 95% CI, 92.2%-93.8%) did not experience an ischemic symptom. Conclusions and Relevance Among patients undergoing noncardiac surgery, peak postoperative hsTnT during the first 3 days after surgery was significantly associated with 30-day mortality. Elevated postoperative hsTnT without an ischemic feature was also associated with 30-day mortality.

High sensitivity troponin T concentrations in patients undergoing noncardiac surgery: A prospective cohort study☆

OBJECTIVES To determine the proportion of noncardiac surgery patients exceeding the published 99th percentile or change criteria with the high sensitivity Troponin T (hs-TnT) assay. DESIGN AND METHODS We measured hs-TnT preoperatively and postoperatively on days 1, 2 and 3 in 325 adults. RESULTS Postoperatively 45% (95% CI: 39-50%) of patients had hs-TnT≥14ng/L and 22% (95% CI:17-26%) had an elevation (≥14ng/L) and change (>85%) in hs-TnT. CONCLUSION Further research is needed to inform the optimal hs-TnT threshold and change in this setting.

Assessment of the European society of cardiology 0-hour/1-hour algorithm to rule-out and rule-in acute myocardial infarction

Background: The new European Society of Cardiology guidelines to rule-in and rule-out acute myocardial infarction (AMI) in the emergency department include a rapid assessment algorithm based on high-sensitivity cardiac troponin and sampling at 0 and 1 hour. Emergency department physicians require high sensitivity to confidently rule-out AMI, whereas cardiologists aim to minimize false-positive results. Methods: High-sensitivity troponin I and T assays were used to measure troponin concentrations in patients presenting with chest-pain symptoms and being investigated for possible acute coronary syndrome at hospitals in New Zealand, Australia, and Canada. AMI outcomes were independently adjudicated by at least 2 physicians. The European Society of Cardiology algorithm performance with each assay was assessed by the sensitivity and proportion with AMI ruled out and the positive predictive value and proportion ruled-in. Results: There were 2222 patients with serial high-sensitivity troponin T and high-sensitivity troponin I measurements. The high-sensitivity troponin T algorithm ruled out 1425 (64.1%) with a sensitivity of 97.1% (95% confidence interval [CI], 94.0%–98.8%) and ruled-in 292 (13.1%) with a positive predictive value of 63.4% (95% CI, 57.5%–68.9%). The high-sensitivity troponin I algorithm ruled out 1205 (54.2%) with a sensitivity of 98.8% (95% CI, 96.4%–99.7%)) and ruled-in 310 (14.0%) with a positive predictive value of 68.1% (95% CI, 62.6%–73.2%). Conclusions: The sensitivity of the European Society of Cardiology rapid assessment 0-/1-hour algorithm to rule-out AMI with high-sensitivity troponin may be insufficient for some emergency department physicians to confidently send patients home. These algorithms may prove useful to identify patients requiring expedited management. However, the positive predictive value was modest for both algorithms.

Increasing Cardiac Troponin Changes Measured by a Research High-Sensitivity Troponin I Assay: Absolute vs Percentage Changes and Long-Term Outcomes in a Chest Pain Cohort

To the Editor: With novel high-sensitivity cardiac troponin assays, there is great interest in determining the change needed between successive cardiac troponin measurements to inform the diagnosis of acute myocardial infarction (MI)1 (1). Recent publications have reported both short- and long-term reference change values (RCVs) in healthy populations for both high-sensitivity cardiac troponin I (hs-cTnI) and high-sensitivity cardiac troponin T (hs-cTnT); thus, the RCV perhaps could be the metric used for determining the optimal clinical change value (2, 3). The RCVs for the hs-cTnT and hs-cTnI assays, however, are markedly different, possibly because of assay characteristics, differences in the biology of the 2 isoforms, or the healthy populations studied (1). The optimal clinical change for detecting patients with acute MI might be greater than the published RCVs. Two recent studies that used ROC curve analyses suggest exactly that. Giannitsis et al. (4) determined the optimal δ to be ≥243% for hs-cTnT, whereas we identified an optimal δ of >235% on the basis of MI or death at 30 days for a research hs-cTnI assay (5). Percentages may work well when values are low but may become impractical when more substantial increases occur. The 2 studies neither reported δ in terms of absolute concentration nor defined its role in long-term risk stratification. …

Validation of presentation and 3 h high-sensitivity troponin to rule-in and rule-out acute myocardial infarction

Objective International guidelines to rule-in acute myocardial infarction (AMI) in patients presenting with chest pain to the emergency department (ED) recommend an algorithm using high-sensitivity cardiac troponin (hs-cTn) sampling on presentation and 3 h following presentation. We tested the diagnostic accuracy of this algorithm by pooling data from five distinct cohorts from three countries of prospectively recruited patients with independently adjudicated outcomes. Method We measured high-sensitivity cardiac troponin I (hs-cTnI) and high-sensitivity cardiac troponin T (hs-cTnT) on presentation (0 h) and 3 h post-presentation samples in adult patients attending an ED with possible AMI to validate the European Society of Cardiology (ESC) Working Group on Acute Cardiac Care rule-in algorithm (ESC-rule-in). Specifically, (i) in patients with a 0 h hs-cTn concentration ≤99th percentile and a 3 h hs-cTn >99th percentile, positive patients are those with an absolute change in troponin ≥50% of the 99th percentile, and (ii) in patients with a 0 and 3 h hs-cTn >99th percentile, positive patients are those with a relative change in troponin of ≥20%. We concurrently assessed the efficacy of the 0 and 3 h hs-cTn <99th percentile to rule-out AMI. Results 1061 patients with hs-cTnI and 985 with hs-cTnT were included. The ESC-rule-in positive predictive value (PPV) was 83.5% (95% CI 74.9% to 90.1%) for hs-cTnI and 72.0% (95% CI 62.1% to 80.5%) for hs-cTnT. Forty-six AMIs (34.9%) were not ruled in using hs-cTnI and 62 (46.2%) using hs-cTnT. The sensitivity of the 99th percentile to rule-out AMI was 93.2% (95% CI 87.5% to 96.8%) for hs-cTnI and 94.8% (95% CI 89.5% to 97.9%) for hs-cTnT. Conclusions The ESC-rule-in algorithm has good PPV with hs-cTnI and reasonable with hs-cTnT and can rule-in over 50% of AMIs. However, the sensitivity of the 99th percentile to rule-out AMI is too low for clinical use.