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1.  Fasting Levels of High-Sensitivity Growth Hormone Predict Cardiovascular Morbidity and Mortality 
Both pathological excess and deficiency of growth hormone (GH) are associated with cardiovascular mortality.
The goal of this study was to test whether fasting levels of growth hormone measured with a high-sensitivity assay (hs-GH) predict cardiovascular morbidity and mortality at the population level.
We studied 4,323 participants (age 46 to 68 years; mean age 58 years; 59% women) of the Swedish, population-based Malmö Diet and Cancer study examined in 1991 to 1994. Using multivariate-adjusted Cox proportional hazards models, we related baseline levels of fasting hs-GH to incidence of coronary artery disease, stroke, congestive heart failure, all-cause mortality, and cardiovascular mortality.
During a median follow-up of 16.2 years, hs-GH (hazard ratio [HR]/SD increment of natural logarithm of fasting hs-GH) was independently associated with increased risk of coronary artery disease (397 events; HR: 1.11; 95% confidence interval [CI]: 1.01 to 1.23; p = 0.04), stroke (251 events; HR: 1.18; 95% CI: 1.04 to 1.34; p = 0.01), congestive heart failure (107 events; HR: 1.25; 95% CI: 1.03 to 1.52; p = 0.02), all-cause mortality (645 events; HR: 1.17; 95% CI: 1.08 to 1.26; p < 0.001) and cardiovascular mortality (186 events; HR: 1.43; 95% CI: 1.24 to 1.66; p < 0.001). The addition of hs-GH to a model with conventional cardiovascular risk factors significantly reclassified risk, with a category-free net reclassification improvement (>0) of 0.542 (95% CI: 0.205 to 0.840) in cardiovascular mortality.
Higher values of hs-GH were associated with an increased risk of cardiovascular morbidity and mortality.
PMCID: PMC4180127  PMID: 25277616
cardiovascular disease; epidemiology; growth hormone; mortality; CAD, coronary artery disease; CHF, congestive heart failure; GH, growth hormone; GHD, growth hormone deficiency; HDL-C, high-density lipoprotein cholesterol; HR, hazard ratio; hs-GH, high-sensitivity growth hormone; LDL-C, low-density lipoprotein cholesterol; NRI, net reclassification improvement; NT-proBNP, N-terminal pro-brain natriuretic peptide
2.  The Role of Apoptosis-Induced Proliferation for Regeneration and Cancer 
Genes dedicated to killing cells must have evolved because of their positive effects on organismal survival. Positive functions of apoptotic genes have been well established in a large number of biological contexts, including their role in eliminating damaged and potentially cancerous cells. More recently, evidence has suggested that proapoptotic proteins—mostly caspases—can induce proliferation of neighboring surviving cells to replace dying cells. This process, that we will refer to as “apoptosis-induced proliferation,” may be critical for stem cell activity and tissue regeneration. Depending on the caspases involved, at least two distinct types of apoptosis-induced proliferation can be distinguished. One of these types have been studied using a model in which cells have initiated cell death, but are prevented from executing it because of effector caspase inhibition, thereby generating “undead” cells that emit persistent mitogen signaling and overgrowth. Such conditions are likely to contribute to certain forms of cancer. In this review, we summarize the current knowledge of apoptosis-induced proliferation and discuss its relevance for tissue regeneration and cancer.
Proapoptotic proteins (e.g., caspases) from dying cells can induce proliferation of neighboring surviving cells. This allows tissues to replace damaged cells with the progeny of healthy neighbors.
PMCID: PMC3405855  PMID: 22855725
3.  The Role of Apoptosis-Induced Proliferation for Regeneration and Cancer 
Genes dedicated to killing cells must have evolved because of their positive effects on organismal survival. Positive functions of apoptotic genes have been well established in a large number of biological contexts, including their role in eliminating damaged and potentially cancerous cells. More recently, evidence has suggested that proapoptotic proteins—mostly caspases—can induce proliferation of neighboring surviving cells to replace dying cells. This process, that we will refer to as “apoptosis-induced proliferation,” may be critical for stem cell activity and tissue regeneration. Depending on the caspases involved, at least two distinct types of apoptosis-induced proliferation can be distinguished. One of these types have been studied using a model in which cells have initiated cell death, but are prevented from executing it because of effector caspase inhibition, thereby generating “undead” cells that emit persistent mitogen signaling and overgrowth. Such conditions are likely to contribute to certain forms of cancer. In this review, we summarize the current knowledge of apoptosis-induced proliferation and discuss its relevance for tissue regeneration and cancer.
PMCID: PMC3405855  PMID: 22855725
4.  UTX coordinates steroid hormone-mediated autophagy and cell death 
Nature communications  2013;4:2916.
Correct spatial and temporal induction of numerous cell type-specific genes during development requires regulated removal of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification. Here we show that the H3K27me3 demethylase dUTX is required for hormone-mediated transcriptional regulation of apoptosis and autophagy genes during ecdysone-regulated programmed cell death of Drosophila salivary glands. We demonstrate that dUTX binds to the nuclear hormone receptor complex Ecdysone Receptor/Ultraspiracle, and is recruited to the promoters of key apoptosis and autophagy genes. Salivary gland cell death is delayed in dUTX mutants, with reduced caspase activity and autophagy that coincides with decreased apoptosis and autophagy gene transcripts. We further show that salivary gland degradation requires dUTX catalytic activity. Our findings provide evidence for an unanticipated role for UTX demethylase activity in regulating hormone-dependent cell death and demonstrate how a single transcriptional regulator can modulate a specific complex functional outcome during animal development.
PMCID: PMC3973156  PMID: 24336022
5.  Plasma adrenomedullin is associated with short-term mortality and vasopressor requirement in patients admitted with sepsis 
Critical Care  2014;18(1):R34.
The incidence of death among patients admitted for severe sepsis or septic shock is high. Adrenomedullin (ADM) plays a central role in initiating the hyperdynamic response during the early stages of sepsis. Pilot studies indicate an association of plasma ADM with the severity of the disease. In the present study we utilized a novel sandwich immunoassay of bioactive plasma ADM in patients hospitalized with sepsis in order to assess the clinical utility.
We enrolled 101 consecutive patients admitted to the emergency department with suspected sepsis in this study. Sepsis was defined by fulfillment of at least two systemic inflammatory response syndrome (SIRS) criteria plus clinical suspicion of infection. Plasma samples for ADM measurement were obtained on admission and for the next four days. The 28-day mortality rate was recorded.
ADM at admission was associated with severity of disease (correlation with Acute Physiology and Chronic Health Evaluation II (APACHE II) score: r = 0.46; P <0.0001). ADM was also associated with 28-day mortality (ADM median (IQR): survivors: 50 (31 to 77) pg/mL; non-survivors: 84 (48 to 232) pg/mL; P <0.001) and was independent from and additive to APACHE II (P = 0.02). Cox regression analysis revealed an additive value of serial measurement of ADM over baseline assessment for prediction of 28-day mortality (P < 0.01). ADM was negatively correlated with mean arterial pressure (r = -0.39; P <0.0001), and it strongly discriminated those patients requiring vasopressor therapy from the others (ADM median (IQR): no vasopressors 48 (32 to 75) pg/mL; with vasopressors 129 (83 to 264) pg/mL, P <0.0001).
In patients admitted with sepsis, severe sepsis or septic shock plasma ADM is strongly associated with severity of disease, vasopressor requirement and 28-day mortality.
PMCID: PMC4056312  PMID: 24533868
6.  Ligand-independent activation of the Hedgehog pathway displays non-cell autonomous proliferation during eye development in Drosophila 
Mechanisms of development  2012;129(0):98-108.
Deregulation of the Hedgehog (Hh) signaling pathway is associated with the development of human cancer including medullobastoma and basal cell carcinoma. Loss of Patched or activation of Smoothened in mouse models increases the occurrence of tumors. Likewise, in a Drosophila eye model, deregulated Hedgehog signaling causes overgrowth of eye and head tissues. Surprisingly, we show that cells with deregulated Hh signaling do not or only little contribute to the tissue overgrowth. Instead, they become more sensitive to apoptosis and may eventually be eliminated. Nevertheless, these mutant cells increase proliferation in the adjacent wild-type tissue, i.e., in a non-cell autonomous manner. This non-cell autonomous effect is position-dependent and restricted to mutant cells in the anterior portion of the eye. We also observe precocious non-cell autonomous differentiation in genetic mosaics with deregulated Hh signaling. Together, these non-cell autonomous growth and differentiation phenotypes in the Drosophila eye model reveal another strategy by which oncogenes may generate a supportive micro-environment for tumor growth.
PMCID: PMC3917606  PMID: 22677792
Hedgehog signaling; Costal-2; Patched; Non-cell autonomous overgrowth
7.  Genetic Models of Apoptosis-Induced Proliferation Decipher Activation of JNK and Identify a Requirement of EGFR Signaling for Tissue Regenerative Responses in Drosophila 
PLoS Genetics  2014;10(1):e1004131.
Recent work in several model organisms has revealed that apoptotic cells are able to stimulate neighboring surviving cells to undergo additional proliferation, a phenomenon termed apoptosis-induced proliferation. This process depends critically on apoptotic caspases such as Dronc, the Caspase-9 ortholog in Drosophila, and may have important implications for tumorigenesis. While it is known that Dronc can induce the activity of Jun N-terminal kinase (JNK) for apoptosis-induced proliferation, the mechanistic details of this activation are largely unknown. It is also controversial if JNK activity occurs in dying or in surviving cells. Signaling molecules of the Wnt and BMP families have been implicated in apoptosis-induced proliferation, but it is unclear if they are the only ones. To address these questions, we have developed an efficient assay for screening and identification of genes that regulate or mediate apoptosis-induced proliferation. We have identified a subset of genes acting upstream of JNK activity including Rho1. We also demonstrate that JNK activation occurs both in apoptotic cells as well as in neighboring surviving cells. In a genetic screen, we identified signaling by the EGFR pathway as important for apoptosis-induced proliferation acting downstream of JNK signaling. These data underscore the importance of genetic screening and promise an improved understanding of the mechanisms of apoptosis-induced proliferation.
Author Summary
Work in recent years has revealed that apoptotic caspases not only induce apoptosis, but also have non-apoptotic functions. One of these functions is apoptosis-induced proliferation, a relatively recently discovered phenomenon by which apoptotic cells induce proliferation of surviving neighboring cells. This phenomenon may have important implications for stem cell activity, tissue regeneration and tumorigenesis. Here, we describe the development of a genetic model of apoptosis-induced proliferation and the use of this model for convenient and unbiased genetic screening to identify genes involved in the process. We tested mutants of our RNAi transgenic lines targeting the core components of the apoptotic pathway and of JNK signaling, a known mediator of apoptosis-induced proliferation. These assays demonstrate the feasibility of the system for systematic genetic screening and identified several new genes upstream of JNK that are involved in apoptosis-induced proliferation. Finally, we tested the model in a pilot screen for chromosome arm 2L and identified spi, the EGF ligand in flies, as important for apoptosis-induced proliferation. We confirmed the involvement of EGF in a genuine apoptosis-induced regeneration system. These data underscore the importance of genetic screening and promise an improved understanding of the mechanisms of apoptosis-induced proliferation and regeneration.
PMCID: PMC3907308  PMID: 24497843
8.  Plasma copeptin and the risk of diabetes mellitus 
Circulation  2010;121(19):2102-2108.
Animal studies suggest that the arginine vasopressin (AVP) system may play a role in glucose metabolism, but data from humans are limited.
Methods and Results
We analysed plasma copeptin (copeptin), a stable C-terminal fragment of the AVP pro-hormone. Using baseline and longitudinal data from a Swedish population-based sample (n=4742, mean age 58 years, 60% women), we examined the association of increasing quartiles of copeptin (lowest quartile as reference) with prevalent diabetes at baseline, insulin resistance (top quartile of fasting plasma insulin among non-diabetic subjects), and incident diabetes on long-term follow up using multivariable logistic regression. New-onset diabetes was ascertained through 3 national and regional registers. All models were adjusted for clinical and anthropometric risk factors, cystatin C, and C-reactive protein. In cross-sectional analyses, increasing copeptin was associated with prevalent diabetes (P=0.04) and insulin resistance (P<0.001). During 12.6 years of follow up 174 subjects (4%) developed new-onset diabetes. The odds of developing diabetes increased across increasing quartiles of copeptin, even after additional adjustment for baseline fasting glucose and insulin (adjusted odds ratios 1.0, 1.37, 1.79, and 2.09; P for trend =0.004). The association with incident diabetes remained significant in analyses restricted to subjects with fasting whole blood glucose <5.4 mmol/L at baseline (adjusted odds ratios 1.0, 1.80, 1.92, and 3.48; P=0.001).
Elevated copeptin predicts increased risk for diabetes, independent of established clinical risk factors, including fasting glucose and insulin. These findings could have implications for risk assessment, novel anti-diabetic treatments, and metabolic side effects from AVP system modulation.
PMCID: PMC3763235  PMID: 20439785
arginine vasopressin; copeptin; diabetes mellitus; risk factors; epidemiology
9.  De-Regulation of JNK and JAK/STAT Signaling in ESCRT-II Mutant Tissues Cooperatively Contributes to Neoplastic Tumorigenesis 
PLoS ONE  2013;8(2):e56021.
Multiple genes involved in endocytosis and endosomal protein trafficking in Drosophila have been shown to function as neoplastic tumor suppressor genes (nTSGs), including Endosomal Sorting Complex Required for Transport-II (ESCRT-II) components vacuolar protein sorting 22 (vps22), vps25, and vps36. However, most studies of endocytic nTSGs have been done in mosaic tissues containing both mutant and non-mutant populations of cells, and interactions among mutant and non-mutant cells greatly influence the final phenotype. Thus, the true autonomous phenotype of tissues mutant for endocytic nTSGs remains unclear. Here, we show that tissues predominantly mutant for ESCRT-II components display characteristics of neoplastic transformation and then undergo apoptosis. These neoplastic tissues show upregulation of c-Jun N-terminal Kinase (JNK), Notch, and Janus Kinase (JAK)/Signal Transducer and Activator of Transcription (STAT) signaling. Significantly, while inhibition of JNK signaling in mutant tissues partially inhibits proliferation, inhibition of JAK/STAT signaling rescues other aspects of the neoplastic phenotype. This is the first rigorous study of tissues predominantly mutant for endocytic nTSGs and provides clear evidence for cooperation among de-regulated signaling pathways leading to tumorigenesis.
PMCID: PMC3572140  PMID: 23418496
10.  Association of common variants in NPPA and NPPB with circulating natriuretic peptides and blood pressure 
Nature genetics  2009;41(3):348-353.
We examined the association of common variants at the NPPA-NPPB locus with circulating concentrations of the natriuretic peptides, which have blood pressure–lowering properties. We genotyped SNPs at the NPPA-NPPB locus in 14,743 individuals of European ancestry, and identified associations of plasma atrial natriuretic peptide with rs5068 (P = 8 × 10−70), rs198358 (P = 8 × 10−30) and rs632793 (P = 2 × 10−10), and of plasma B-type natriuretic peptide with rs5068 (P = 3 × 10−12), rs198358 (P = 1 × 10−25) and rs632793 (P = 2 × 10−68). In 29,717 individuals, the alleles of rs5068 and rs198358 that showed association with increased circulating natriuretic peptide concentrations were also found to be associated with lower systolic (P = 2 × 10−6 and 6 × 10−5, respectively) and diastolic blood pressure (P = 1 × 10−6 and 5 × 10−5), as well as reduced odds of hypertension (OR = 0.85, 95% CI = 0.79–0.92, P = 4 × 10−5; OR = 0.90, 95% CI = 0.85–0.95, P = 2 × 10−4, respectively). Common genetic variants at the NPPA-NPPB locus found to be associated with circulating natriuretic peptide concentrations contribute to interindividual variation in blood pressure and hypertension.
PMCID: PMC2664511  PMID: 19219041
11.  Notch Signaling Activates Yorkie Non-Cell Autonomously in Drosophila 
PLoS ONE  2012;7(6):e37615.
In Drosophila imaginal epithelia, cells mutant for the endocytic neoplastic tumor suppressor gene vps25 stimulate nearby untransformed cells to express Drosophila Inhibitor-of-Apoptosis-Protein-1 (DIAP-1), conferring resistance to apoptosis non-cell autonomously. Here, we show that the non-cell autonomous induction of DIAP-1 is mediated by Yorkie, the conserved downstream effector of Hippo signaling. The non-cell autonomous induction of Yorkie is due to Notch signaling from vps25 mutant cells. Moreover, activated Notch in normal cells is sufficient to induce non-cell autonomous Yorkie activity in wing imaginal discs. Our data identify a novel mechanism by which Notch promotes cell survival non-cell autonomously and by which neoplastic tumor cells generate a supportive microenvironment for tumor growth.
PMCID: PMC3367968  PMID: 22679484
12.  Use of procalcitonin for the diagnosis of pneumonia in patients presenting with a chief complaint of dyspnoea: results from the BACH (Biomarkers in Acute Heart Failure) trial 
European Journal of Heart Failure  2012;14(3):278-286.
Biomarkers have proven their ability in the evaluation of cardiopulmonary diseases. We investigated the utility of concentrations of the biomarker procalcitonin (PCT) alone and with clinical variables for the diagnosis of pneumonia in patients presenting to emergency departments (EDs) with a chief complaint of shortness of breath.
Methods and results
The BACH trial was a prospective, international, study of 1641 patients presenting to EDs with dyspnoea. Blood samples were analysed for PCT and other biomarkers. Relevant clinical data were also captured. Patient outcomes were assessed at 90 days. The diagnosis of pneumonia was made using strictly validated guidelines. A model using PCT was more accurate [area under the curve (AUC) 72.3%] than any other individual clinical variable for the diagnosis of pneumonia in all patients, in those with obstructive lung disease, and in those with acute heart failure (AHF). Combining physician estimates of the probability of pneumonia with PCT values increased the accuracy to >86% for the diagnosis of pneumonia in all patients. Patients with a diagnosis of AHF and an elevated PCT concentration (>0.21 ng/mL) had a worse outcome if not treated with antibiotics (P = 0.046), while patients with low PCT values (<0.05 ng/mL) had a better outcome if they did not receive antibiotic therapy (P = 0.049).
Procalcitonin may aid in the diagnosis of pneumonia, particularly in cases with high diagnostic uncertainty. Importantly, PCT may aid in the decision to administer antibiotic therapy to patients presenting with AHF in which clinical uncertainty exists regarding a superimposed bacterial infection.
Trial registration: NCT00537628
PMCID: PMC3284113  PMID: 22302662
Acute heart failure; Procalcitonin; Pneumonia; Survival; Diagnosis
13.  Serial measurements of midregion proANP and copeptin in ambulatory patients with heart failure: incremental prognostic value of novel biomarkers in heart failure 
Heart  2011;98(5):389-394.
Disease progression in heart failure (HF) reflects derangements in neurohormonal systems, and biomarkers of these systems can help to establish the diagnosis and assess the prognosis. Serial measurements of the precursor peptides of the natriuretic and vasopressin systems (midregional proatrial natriuretic peptide (MR-proANP) and C-terminal provasopressin (copeptin), respectively) should add incremental value to risk stratification in ambulatory patients with HF.
Methods and results
A cohort of 187 patients with class III–IV HF was prospectively enrolled, with biomarkers collected every 3 months over 2 years and analysed in relation to death/transplantation. Time-dependent analyses (dichotomous and continuous variables) showed that increases in MR-proANP (HR 7.6, 95% CI 1.85 to 31.15, p<0.01) and copeptin (HR 2.7, 95% CI 1.27 to 5.61, p=0.01) were associated with increased risk, but, in multivariate analysis adjusted for troponin T (cTnT) ≥0.01 ng/ml, only raised MR-proANP remained an independent predictor (HR 5.49, 95% CI 1.31 to 23.01, p=0.02). Combined increases in MR-proANP and copeptin (HR 9.01, 95% CI 1.24 to 65.26, p=0.03) with cTnT (HR 11.1, 95% CI 1.52 to 80.85, p=0.02), and increases ≥30% above already raised values identified the patients at greatest risk (MR-proANP: HR 10.1, 95% CI 2.34 to 43.38, p=0.002; copeptin: HR 11.5, 95% CI 2.74 to 48.08, p<0.001).
A strategy of serial monitoring of MR-proANP and, of lesser impact, copeptin, combined with cTnT, may be advantageous in detecting and managing the highest-risk outpatients with HF.
PMCID: PMC3276775  PMID: 22194151
MR-pro-ANP; CT-pro-vasopressin (copeptin); chronic heart failure; outcomes; heart failure; heart failure treatment; acute coronary syndrome; acute myocarditis; coronary heart disease; natriuretic peptides; troponin t; acute myocardial infarction
14.  Assessment of conventional cardiovascular risk factors and multiple biomarkers for the prediction of incident heart failure and atrial fibrillation 
To assess the predictive accuracy of conventional cardiovascular risk factors for incident heart failure(HF) and atrial fibrillation(AF) and the added benefit of multiple biomarkers reflecting diverse pathophysiological pathways.
HF and AF are interrelated cardiac diseases associated with substantial morbidity and mortality and increasing incidence. Data on prediction and prevention of these diseases in healthy individuals is limited.
In 5,187 individuals from the community-based Malmö Diet and Cancer study, we studied the performance of conventional risk factors and six biomarkers including midregional pro-atrial natriuretic peptide(MR-proANP), N-terminal pro-B-type natriuretic peptide(Nt-proBNP), midregional pro-adrenomedullin, cystatin C, C-reactive protein(CRP) and copeptin.
During a mean follow-up of 14 years,112 individuals were diagnosed with HF and 284 individuals with AF. Nt-proBNP(HR=1.63 per SD,95%CI=1.29–2.06,p<0.001), CRP(HR=1.57 per SD,95%CI=1.28–1.94,p<0.001) and MR-proANP(HR=1.26 per SD,95%CI=1.02-1-56,p=0.03) predicted incident HF independently of conventional risk factors and other biomarkers. MR-proANP(HR=1.62,95%CI=1.42-1.84,p<0.001) and CRP(HR=1.18,95%CI=1.03–1.34,p=0.01) independently predicted AF. Addition of biomarkers to conventional risk factors improved C-statistics from 0.815 to 0.842 for HF and from 0.732 to 0.753 for AF and the Integrated discriminatory index for both diseases(p<0.001). Net reclassification improvement with biomarkers was observed in 22% of individuals for HF(NRI,p<0.001) and in 7% for AF(NRI,p=0.06), mainly due to up-classification of individuals who developed disease(HF:29%,AF:19%). Addition of CRP to natriuretic peptides did not improve discrimination or reclassification.
Conventional cardiovascular risk factors predict incident HF and AF with reasonable accuracy in middle-aged individuals free from disease. Natriuretic peptides, but not other biomarkers, improve discrimination modestly for both diseases above and beyond conventional risk factors and substantially improve classification for HF.
PMCID: PMC3005324  PMID: 21070922
Atrial Fibrillation; Heart failure; Prediction; Natriuretic peptides; Risk factors; Epidemiology
15.  Apoptosis, Stem Cells, and Tissue Regeneration 
Science signaling  2010;3(145):re8-.
Most metazoans have at least some ability to regenerate damaged cells and tissues, although the regenerative capacity varies depending on the species, organ, or developmental stage. Cell replacement and regeneration occur in two contexts: renewal of spent cells during tissue homeostasis (homeostatic growth), and in response to external injury, wounding, or amputation (epimorphic regeneration). Model organisms that display remarkable regenerative capacity include amphibians, planarians, Hydra, and the vertebrate liver. In addition, several mammalian organs—including the skin, gut, kidney, muscle, and even the human nervous system—have some ability to replace spent or damaged cells. Although the regenerative response is complex, it typically involves the induction of new cell proliferation through formation of a blastema, followed by cell specification, differentiation, and patterning. Stem cells and undifferentiated progenitor cells play an important role in both tissue homeostasis and tissue regeneration. Stem cells are typically quiescent or passing slowly through the cell cycle in adult tissues, but they can be activated in response to cell loss and wounding. A series of studies, mostly performed in Drosophila as well as in Hydra, Xenopus, and mouse, has revealed an unexpected role of apoptotic caspases in the production of mitogenic signals that stimulate the proliferation of stem and progenitor cells to aid in tissue regeneration. This Review summarizes some of the key findings and discusses links to stem cell biology and cancer.
PMCID: PMC2991142  PMID: 20978240
16.  Drosophila IAP1-Mediated Ubiquitylation Controls Activation of the Initiator Caspase DRONC Independent of Protein Degradation 
PLoS Genetics  2011;7(9):e1002261.
Ubiquitylation targets proteins for proteasome-mediated degradation and plays important roles in many biological processes including apoptosis. However, non-proteolytic functions of ubiquitylation are also known. In Drosophila, the inhibitor of apoptosis protein 1 (DIAP1) is known to ubiquitylate the initiator caspase DRONC in vitro. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by caspase inhibition (“undead” cells), it is thought that DIAP1-mediated ubiquitylation causes proteasomal degradation of DRONC, protecting cells from apoptosis. However, contrary to this model, we show here that DIAP1-mediated ubiquitylation does not trigger proteasomal degradation of full-length DRONC, but serves a non-proteolytic function. Our data suggest that DIAP1-mediated ubiquitylation blocks processing and activation of DRONC. Interestingly, while full-length DRONC is not subject to DIAP1-induced degradation, once it is processed and activated it has reduced protein stability. Finally, we show that DRONC protein accumulates in “undead” cells due to increased transcription of dronc in these cells. These data refine current models of caspase regulation by IAPs.
Author Summary
The Drosophila inhibitor of apoptosis 1 (DIAP1) readily promotes ubiquitylation of the CASPASE-9–like initiator caspase DRONC in vitro and in vivo. Because DRONC protein accumulates in diap1 mutant cells that are kept alive by effector caspase inhibition—producing so-called “undead” cells—it has been proposed that DIAP1-mediated ubiquitylation would target full-length DRONC for proteasomal degradation, ensuring survival of normal cells. However, this has never been tested rigorously in vivo. By examining loss and gain of diap1 function, we show that DIAP1-mediated ubiquitylation does not trigger degradation of full-length DRONC. Our analysis demonstrates that DIAP1-mediated ubiquitylation controls DRONC processing and activation in a non-proteolytic manner. Interestingly, once DRONC is processed and activated, it has reduced protein stability. We also demonstrate that “undead” cells induce transcription of dronc, explaining increased protein levels of DRONC in these cells. This study re-defines the mechanism by which IAP-mediated ubiquitylation regulates caspase activity.
PMCID: PMC3164697  PMID: 21909282
17.  Regulation of EGFR and Notch signaling by distinct isoforms of D-cbl during Drosophila development 
Developmental biology  2010;342(1):1-10.
Cells receive and interpret extracellular signals to regulate cellular responses such as proliferation, cell survival and differentiation. However, proper inactivation of these signals is critical for appropriate homeostasis. Cbl proteins are E3-ubiquitin ligases that restrict receptor tyrosine kinase (RTK) signaling, most notably EGFR (Epidermal Growth Factor Receptor), via the endocytic pathway. Consistently, many mutant phenotypes of Drosophila cbl (D-cbl) are due to inappropriate activation of EGFR signaling. However, not all D-cbl phenotypes can be explained by increased EGFR activity. Here, we report that D-Cbl also negatively regulates Notch activity during eye and wing development. D-cbl produces two isoforms by alternative splicing. The long isoform, D-CblL, regulates the EGFR. We found that the short isoform, D-CblS, preferentially restricts Notch signaling. Specifically, our data imply that D-CblS controls the activity of the Notch ligand Delta. Taken together, these data suggest that D-Cbl controls the EGFR and Notch/Delta signaling pathways through production of two alternatively spliced isoforms during development in Drosophila.
PMCID: PMC2866751  PMID: 20302857
Drosophila; cbl; D-cbl; RTK signaling; EGFR; Notch; Delta
18.  Novel and conventional biomarkers for the prediction of incident cardiovascular events in the community 
Prior studies have conflicted regarding how much information novel biomarkers add to cardiovascular risk assessment.
To evaluate the utility of biomarkers for predicting cardiovascular risk when added to conventional risk factors, using contemporary biomarkers and newer statistical approaches.
Design, Setting, Participants
Between 1991 and 1994, 5067 participants (mean age 58, 60% women) without cardiovascular disease from a prospective cohort in Malmö, Sweden underwent measurement of C-reactive protein (CRP), mid-regional-pro-atrial natriuretic peptide, N-terminal pro-B-type natriuretic peptide (N-BNP), mid-regional-pro-adrenomedullin (MR-proADM), lipoprotein-associated phospholipase-2, and cystatin C. Participants were followed until 2006. First cardiovascular events (myocardial infarction, stroke, coronary death) were ascertained using the Swedish national hospital discharge and cause-of-death registers. Low-, intermediate-, and high-risk were defined as 10-year risks of <6%, 6–19%, or ≥20%, respectively.
Main Outcome Measures
Incident cardiovascular and coronary events.
During median follow-up of 12.8 years, there were 418 cardiovascular and 230 coronary events. Models with conventional risk factors had c-statistics of 0.758 (95% confidence interval [CI], 0.734–0.781) and 0.760 (0.730–0.789) for cardiovascular and coronary events. Biomarkers retained in backward-elimination models were N-BNP and CRP for cardiovascular events, and N-BNP and MR-proADM for coronary events, which raised the c-statistic by 0.007 (p=0.04) and 0.009 (p=0.08), respectively. The proportion of participants reclassified was modest (8% for cardiovascular risk, 5% for coronary risk). The net reclassification improvement (NRI) was non-significant for cardiovascular events (0.0%, 95%CI, −4.3%–4.3%) and coronary events (4.7%, −0.76%–10.1%). Greater improvements were observed in analyses restricted to intermediate-risk individuals (cardiovascular events: 7.4%, 95%CI, 0.7%–14.1% [p=0.03]; coronary events: 14.6%, 5.0%–24.2% [p=0.003]). However, correct re-classification was almost entirely confined to down-classification of individuals without events, rather than up-classification of those with events.
Selected biomarkers may be used to predict future cardiovascular events, but the gains over conventional risk factors are minimal. Risk classification improved in intermediate-risk individuals, mainly through the identification of those unlikely to develop events.
PMCID: PMC3090639  PMID: 19567439
19.  Plasma C-Terminal Pro–Endothelin-1 is Associated with Left Ventricular Mass Index and Aortic Root Diameter in African American Adults with Hypertension 
Journal of human hypertension  2010;25(2):106-113.
Endothelin-1 (ET-1), a circulating vasoactive peptide with a potent vasoconstricting and mitogenic properties, may contribute to target-organ damage in hypertension. A novel assay for the measurement of C-terminal pro-endothelin-1 (CT-proET-1), a stable fragment of ET-1 precursor, has been recently validated as a reliable measure of ET-1 activity in the plasma. We tested the hypothesis that in African American adults with hypertension, plasma CT-proET-1 is associated with left ventricular (LV) mass and aortic root diameter.
Participants included 1041 African Americans (65±9 y, 72% women) with hypertension. Plasma CT-proET-1 was measured by an immunoluminometric assay. LV Mass and aortic root diameter were measured according to the American Society of Echocardiography guidelines, and LV mass was indexed by height to the power 2.7 (LVMi). Multivariable regressions analyses (using generalized estimating equations) were employed to assess whether plasma CT-proET-1 was associated with LVMi and aortic root diameter independent of potential confounding variables.
Plasma CT-proET-1 was significantly correlated with LVMi (r =0.21, P <0.0001) and aortic root diameter (r =0.09, P= 0.004). In separate multivariable regression models that adjusted for age, sex, body mass index, total and high density lipoprotein cholesterol, smoking history, diabetes, previous history of myocardial infarction or stroke, and blood pressure-lowering medication and statin use, log CT-proET-1 was significantly associated with greater LVMi (P =0.001) and greater aortic root diameter (P =0.006).
CT-proET-1 is independently associated with LVMi and aortic root diameter and may be a marker of target-organ damage in African Americans adults with hypertension.
PMCID: PMC2921559  PMID: 20182452
20.  Identification of Chronic Heart Failure Patients with a High 12-Month Mortality Risk Using Biomarkers Including Plasma C-Terminal Pro-Endothelin-1 
PLoS ONE  2011;6(1):e14506.
We hypothesised that assessment of plasma C-terminal pro-endothelin-1 (CT-proET-1), a stable endothelin-1 precursor fragment, is of prognostic value in patients with chronic heart failure (CHF), beyond other prognosticators, including N-terminal pro-B-type natriuretic peptide (NT-proBNP).
We examined 491 patients with systolic CHF (age: 63±11 years, 91% men, New York Heart Association [NYHA] class [I/II/III/IV]: 9%/45%/38%/8%, 69% ischemic etiology). Plasma CT-proET-1 was detected using a chemiluminescence immunoassay.
Increasing CT-proET-1 was a predictor of increased cardiovascular mortality at 12-months of follow-up (standardized hazard ratio 1.42, 95% confidence interval [CI] 1.04–1.95, p = 0.03) after adjusting for NT-proBNP, left ventricular ejection fraction (LVEF), age, creatinine, NYHA class. In receiver operating characteristic curve analysis, areas under curve for 12-month follow-up were similar for CT-proET-1 and NT-proBNP (p = 0.40). Both NT-proBNP and CT-proET-1 added prognostic value to a base model that included LVEF, age, creatinine, and NYHA class. Adding CT-proET-1 to the base model had stronger prognostic power (p<0.01) than adding NT-proBNP (p<0.01). Adding CT-proET-1 to NT-proBNP in this model yielded further prognostic information (p = 0.02).
Plasma CT-proET-1 constitutes a novel predictor of increased 12-month cardiovascular mortality in patients with CHF. High CT-proET-1 together with high NT-proBNP enable to identify patients with CHF and particularly unfavourable outcomes.
PMCID: PMC3022013  PMID: 21264211
21.  Dual roles of Drosophila p53 in cell death and cell differentiation 
Cell death and differentiation  2009;17(6):912-921.
The mammalian p53-family consists of p53, p63 and p73. While p53 accounts for tumor suppression through cell cycle arrest and apoptosis, the functions of p63 and p73 are more diverse and also include control of cell differentiation. The Drosophila genome contains only one p53 homolog, Dp53. Previous work has established that Dp53 induces apoptosis, but not cell cycle arrest. Here, by using the developing eye as a model, we show that Dp53-induced apoptosis is primarily dependent on the pro-apoptotic gene hid, but not reaper, and occurs through the canonical apoptosis pathway. Importantly, similar to p63 and p73, expression of Dp53 also inhibits cellular differentiation of photoreceptor neurons and cone cells in the eye independently of its apoptotic function. Intriguingly, expression of the human cell cycle inhibitor p21 or its Drosophila homolog dacapo can suppress both Dp53-induced cell death and differentiation defects in Drosophila eyes. These findings provide new insights into the pathways activated by Dp53 and reveal that Dp53 incorporates functions of multiple p53-family members.
PMCID: PMC3014827  PMID: 19960025
22.  Relation of Natriuretic Peptides and Midregional Proadrenomedullin to Cardiac Chamber Volumes by Computed Tomography in Patients without Heart Failure: From the ROMICAT Trial 
Clinical chemistry  2010;56(4):651-660.
Stress myocyte biomarkers are used prognostically in patients with cardiovascular disease. We examined associations between amino-terminal pro–B-type natriuretic peptide (NT-proBNP), midregional pro–A-type natriuretic peptide (MR-proANP), and midregional proadrenomedullin (MR-proADM) concentrations and cardiac chamber volumes in chest pain patients without heart failure by use of computed tomography (CT).
At the time of 64-slice CT scan, we acquired plasma and serum samples for these biomarkers from 346 patients [mean (SD) age 53 (12) years, 65% men]. Left atrial volume (LAV) and left ventricular volumes at end-diastole (LVEDV) and end-systole (LVESV) were measured and indexed to body surface area (LAVI, LVEDI, LVESI).
Concentrations of both natriuretic peptides were correlated with LAV and LAVI (r=0.19–0.32, all P ≤ 0.0005) and MR-proADM with LV volumes and indices (r=−0.14 to −0.21, all P ≤ 0.01). NT-proBNP and MR-proANP concentrations were higher in the top quartiles of patients than the lowest quartiles using LAV and LAVI, whereas MR-proADM concentrations were lower in the top quartiles of LV measures. In adjusted analyses, patients had 2- to 4-fold increased risk of LA enlargement for every incremental increase in log10NT-proBNP [LAV odds ratio (OR) 2.4, P = 0.03; LAVI OR 4.0, P = 0.003] and 10- to 13-fold increased risk of LA enlargement for every incremental increase in log10MR-proANP (LAV OR 10.7, P = 0.009; LAVI OR 13.1, P = 0.004).
In patients without heart failure, both NT-proBNP and MR-proANP concentrations are independently associated with LA enlargement, whereas MR-proADM concentrations are correlated with LV volumes. This may partially explain the well-recognized value of natriuretic peptides for use in risk stratification.
PMCID: PMC2997388  PMID: 20185624
23.  Copeptin and risk stratification in patients with acute dyspnea 
Critical Care  2010;14(6):R213.
The identification of patients at highest risk for adverse outcome who are presenting with acute dyspnea to the emergency department remains a challenge. This study investigates the prognostic value of Copeptin, the C-terminal part of the vasopressin prohormone alone and combined to N-terminal pro B-type natriuretic peptide (NT-proBNP) in patients with acute dyspnea.
We conducted a prospective, observational cohort study in the emergency department of a university hospital and enrolled 287 patients with acute dyspnea.
Copeptin levels were elevated in non-survivors (n = 29) compared to survivors at 30 days (108 pmol/l, interquartile range (IQR) 37 to 197 pmol/l) vs. 18 pmol/l, IQR 7 to 43 pmol/l; P < 0.0001). The areas under the receiver operating characteristic curve (AUC) to predict 30-day mortality were 0.83 (95% confidence interval (CI) 0.76 to 0.90), 0.76 (95% CI 0.67 to 0.84) and 0.63 (95% CI 0.53 to 0.74) for Copeptin, NT-proBNP and BNP, respectively (Copeptin vs. NTproBNP P = 0.21; Copeptin vs. BNP P = 0.002). When adjusted for common cardiovascular risk factors and NT-proBNP, Copeptin was the strongest independent predictor for short-term mortality in all patients (HR 3.88 (1.94 to 7.77); P < 0.001) and especially in patients with acute decompensated heart failure (ADHF) (HR 5.99 (2.55 to 14.07); P < 0.0001). With the inclusion of Copeptin to the adjusted model including NTproBNP, the net reclassification improvement (NRI) was 0.37 (P < 0.001). An additional 30% of those who experienced events were reclassified as high risk, and an additional 26% without events were reclassified as low risk.
Copeptin is a new promising prognostic marker for short-term mortality independently and additive to natriuretic peptide levels in patients with acute dyspnea.
PMCID: PMC3220005  PMID: 21106053
24.  The H3K27me3 Demethylase dUTX Is a Suppressor of Notch- and Rb-Dependent Tumors in Drosophila▿  
Molecular and Cellular Biology  2010;30(10):2485-2497.
Trimethylated lysine 27 of histone H3 (H3K27me3) is an epigenetic mark for gene silencing and can be demethylated by the JmjC domain of UTX. Excessive H3K27me3 levels can cause tumorigenesis, but little is known about the mechanisms leading to those cancers. Mutants of the Drosophila H3K27me3 demethylase dUTX display some characteristics of Trithorax group mutants and have increased H3K27me3 levels in vivo. Surprisingly, dUTX mutations also affect H3K4me1 levels in a JmjC-independent manner. We show that a disruption of the JmjC domain of dUTX results in a growth advantage for mutant cells over adjacent wild-type tissue due to increased proliferation. The growth advantage of dUTX mutant tissue is caused, at least in part, by increased Notch activity, demonstrating that dUTX is a Notch antagonist. Furthermore, the inactivation of Retinoblastoma (Rbf in Drosophila) contributes to the growth advantage of dUTX mutant tissue. The excessive activation of Notch in dUTX mutant cells leads to tumor-like growth in an Rbf-dependent manner. In summary, these data suggest that dUTX is a suppressor of Notch- and Rbf-dependent tumors in Drosophila melanogaster and may provide a model for UTX-dependent tumorigenesis in humans.
PMCID: PMC2863695  PMID: 20212086
25.  Plasma C-Terminal Pro-Endothelin-1 Is Associated With Target-Organ Damage in African Americans With Hypertension 
American journal of hypertension  2010;23(11):1204-1208.
Endothelin-1 (ET-1) is a vasoactive peptide with vasoconstrictor and mitogenic properties. We investigated whether plasma levels of C-terminal pro-ET-1 (CT-proET-1), a newly described stable fragment of the ET-1 precursor, are associated with target-organ damage in hypertension.
Participants included 981 African Americans (65 ± 9 years, 71% women) and 812 non-Hispanic whites (61 ± 9 years, 54% women) ascertained from sibships with hypertension. We measured plasma CT-proET-1 by an immunoluminometric assay. Measures of target-organ damage included the ankle-brachial index (ABI) and urinary albumin:creatinine ratio (UACR). Multivariable regressions analyses were employed to assess whether plasma CT-proET-1 levels were independently associated with ABI and UACR.
In hypertensive African Americans, higher plasma levels of CT-proET-1 were significantly associated with lower ABI (P < 0.01) and higher UACR (P < 0.01). After adjustment for age, sex, body mass index, systolic blood pressure (SBP) and diastolic blood pressure (BP), diabetes, serum glucose, insulin use, estimated glomerular filtration rate (eGFR), history of smoking, total and high-density lipoprotein cholesterol, medication use, and previous history of myocardial infarction (MI) or stroke, higher plasma levels of CT-proET-1 remained significantly associated with lower ABI (P < 0.01) and higher UACR (P = 0.02). In non-Hispanic white hypertensives, higher plasma levels of CT-proET-1 were weakly associated with higher UACR (P = 0.02) and with lower ABI (P = 0.07). After adjustment for the relevant covariates, no statistically significant associations between CT-proET-1 and ABI or UACR were present in whites.
Plasma levels of CT-proET-1 were independently associated with lower ABI and greater UACR in African American but not non-Hispanic white adults with hypertension.
PMCID: PMC2957561  PMID: 20634796
ankle-brachial index; blood pressure; endothelin; hypertension; target-organ damage; urinary albumin:creatinine ratio

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