A systematic review and meta-analysis of the effect of routine early angiography in patients with return of spontaneous circulation after Out-of-Hospital Cardiac Arrest.

BACKGROUND: Early coronary angiography (CAG) has been reported in individual studies and systematic reviews to significantly improve outcomes of patients with return of spontaneous circulation (ROSC) after cardiac arrest (CA). METHODS: We undertook a systematic review and meta-analysis to evaluate the impact of early CAG on key clinical outcomes in comatose patients after ROSC following out-of-hospital CA of presumed cardiac origin. We searched the PubMED, EMBASE, CINAHL, ERIC and Cochrane Central Register of Controlled Trials databases from 1990 until April 2020. Eligible studies compared patients undergoing early CAG to patients with late or no CAG. When randomized controlled trials (RCTs) existed for a specific outcome, we used their results to estimate the effect of the intervention. In the absence of randomized data, we used observational data. We excluded studies at high risk of bias according to the Robins-I tool from the meta-analysis. The GRADE system was used to assess certainty of evidence at an outcome level. RESULTS: Of 3738 citations screened, 3 randomized trials and 41 observational studies were eligible for inclusion. Evidence certainty across all outcomes for the RCTs was assessed as low. Randomized data showed no benefit from early as opposed to late CAG across all critical outcomes of survival and survival with favourable neurologic outcome for undifferentiated patients and for patient subgroups without ST-segment-elevation on post ROSC ECG and shockable initial rhythm. CONCLUSION: These results do not support routine early CAG in undifferentiated comatose patients and patients without STE on post ROSC ECG after OHCA. REVIEW REGISTRATION: PROSPERO - CRD42020160152.

Outcomes of out of hospital cardiac arrest in First Nations and non-First Nations patients in Saskatoon.

INTRODUCTION: One in nine (11.7%) people in Saskatchewan identify as First Nations. It is known that First Nations people have a higher burden of cardiovascular disease, but not whether outcomes of out of hospital cardiac arrest are different. METHODS/METHODOLOGY: We reviewed pre-hospital and inpatient records of patients with out of hospital cardiac arrest between January 1st, 2015 and December 31st, 2017. The population consisted of patients aged 18 years or older with out of hospital cardiac arrest of presumed cardiac origin occurring in the catchment area of Saskatoon's emergency medical services (EMS). Variables of interest included age, gender, First Nations status, EMS response times, bystander cardiopulmonary resuscitation (CPR), and shockable rhythm. Outcomes of interest included return of spontaneous circulation (ROSC), survival to hospital admission, and survival to hospital discharge. RESULTS: In all, 372 patients sustained out of hospital cardiac arrest, of which 27 were status First Nations. There were no differences between First Nations and non-First Nations patients in terms of shockable rhythms (24% vs 26%; p = 0.80), ROSC (42% vs 41%; p = 0.87), survival to hospital admission (27% vs 33%; p = 0.53), and survival to hospital discharge (15% vs 12%; p = 0.54). First Nations patients with out of hospital cardiac arrest were significantly younger (mean age 46 vs. 65 years; p < 0.0001) and had shorter EMS response times (median times 5.3 vs. 6.2 min; p = 0.01) when compared to non-First Nations patients. CONCLUSIONS: In Saskatoon, First Nations patients with out of hospital cardiac arrest appear to have similar survival rates when compared with non-First Nations patients. However, First Nations patients sustaining out of hospital cardiac arrest were significantly younger than their non-First Nations counterparts. This highlights a significant public health issue.

RéSUMé: INTRODUCTION: Une personne sur neuf (11,7 %) en Saskatchewan s'identifie comme membre des Premières Nations. On sait que le fardeau des maladies cardiovasculaires est plus lourd pour les membres des Premières Nations, mais on ne sait pas si les résultats d'un arrêt cardiaque en dehors de l'hôpital sont différents. MéTHODES/MéTHODOLOGIE: Nous avons examiné les dossiers pré-hospitaliers et les dossiers des patients hospitalisés ayant subi un arrêt cardiaque hors hôpital entre le 1er janvier 2015 et le 31 décembre 2017. La population était composée de patients âgés de 18 ans ou plus ayant subi un arrêt cardiaque hors de l'hôpital d'origine cardiaque présumée survenu dans la Territoire des services médicaux d'urgence (SMU) de Saskatoon. Les variables d'intérêt comprenaient l'âge, le sexe, le statut des Premières Nations, les temps de réponse des SMU, la réanimation cardiorespiratoire (RCR) des témoins et le rythme choquant. Les résultats d'intérêt comprenaient le retour de la circulation spontanée (RCS), la survie à l'admission à l'hôpital et la survie la sortie de l'hôpital. RéSULTATS : Au total, 372 patients ont subi un arrêt cardiaque hors de l'hôpital, dont 27 étaient des Premières Nations inscrites. Il n'y avait aucune différence entre les patients des Premières Nations et les patients non membres des Premières Nations en termes de rythmes choquants (24 % contre 26 %; p = 0.80), RCS (42% contre 41% ; p = 0.87), survie à l'admission à l'hôpital ( 27% contre 33%; p = 0.53) et la survie à la sortie de l'hôpital (15 % contre 12%; p = 0.54). Les patients des Premières Nations ayant subi un arrêt cardiaque hors hôpital étaient significativement plus jeunes (âge moyen 46 ans contre 65 ans ; p < 0.0001)) et avaient des temps de réponse plus courts aux SMU (temps médian 5,3 contre 6,2 minutes ; p = 0.01) par rapport aux patients des autres nations. CONCLUSIONS: À Saskatoon, les patients des Premières Nations ayant subi un arrêt cardiaque hors de l'hôpital semblent avoir des taux de survie similaires à ceux des patients non autochtones. Cependant, les patients des Premières Nations qui ont subi un arrêt cardiaque hors de l'hôpital étaient beaucoup plus jeunes que leurs homologues des autres nations. Cela met en évidence un problème de santé publique important.

Regulation of epithelial-mesenchymal transition and organoid morphogenesis by a novel TGFß-TCF7L2 isoform-specific signaling pathway.

Alternative splicing contributes to diversification of gene function, yet consequences of splicing on functions of specific gene products is poorly understood. The major transcription factor TCF7L2 undergoes alternative splicing but the biological significance of TCF7L2 isoforms has remained largely to be elucidated. Here, we find that the TCF7L2 E-isoforms maintain, whereas the M and S isoforms disrupt morphogenesis of 3D-epithelial cell-derived organoids via regulation of epithelial-mesenchymal transition (EMT). Remarkably, TCF7L2E2 antagonizes, whereas TCF7L2M2/S2 promotes EMT-like effects in epithelial cells induced by transforming growth factor beta (TGFß) signaling. In addition, we find TGFß signaling reduces the proportion of TCF7L2E to TCF7L2M/S protein in cells undergoing EMT. We also find that TCF7L2 operates via TGFß-Smad3 signaling to regulate EMT. Collectively, our findings unveil novel isoform-specific functions for the major transcription factor TCF7L2 and provide novel links between TCF7L2 and TGFß signaling in the control of EMT-like responses and epithelial tissue morphogenesis.

Impact of Pit-Crew Cardiopulmonary Resuscitation on Out-of-Hospital Cardiac Arrest in Saskatoon.

BACKGROUND: In the prehospital setting, pit-crew models of cardiopulmonary resuscitation (CPR) have shown improvements in survival after out-of-hospital cardiac arrest (OHCA). Certain districts in North America have adopted this model, including Saskatoon, Saskatchewan, Canada. OBJECTIVES: Our objectives were to determine whether pit-crew CPR has an impact on survival to discharge after OHCA in Saskatoon, Canada. METHODS: This was a retrospective pre- and postintervention study. All adult patients who had an OHCA between January 1, 2011 and December 31, 2017 of presumed cardiac origin, in which the resuscitation attempt included CPR by trained prehospital responders, were considered for analysis. Our primary outcome was survival to discharge. Survival to admission and return of spontaneous circulation were secondary outcomes. RESULTS: There were 860 OHCAs considered for our study. After 46 exclusions there were 442 in the non-pit-crew group (average age 63.7 years; 64.5% male) and 372 in the pit-crew group (average age 63.5 years; 67.5% male). Survival to discharge after an OHCA was 10.4% (95% confidence interval 7.7-13.6%) in the non-pit-crew group and 12.4% (95% CI 9.2-16.2%) in the pit-crew group, which did not meet statistical significance. Return of spontaneous circulation and survival to admission were 48.4% and 31.3%, respectively, in the non-pit-crew group and 46.7% and 32.3%, respectively, in the pit-crew group. CONCLUSIONS: In our study, implementation of a pit-crew CPR model was not associated with an improvement in survival to discharge after OHCA.

Emergency department referrals from a provincial medical call centre: Is it more than just 1-800-go-to-emerg?

OBJECTIVE: HealthLine is Saskatchewan's provincial 24-hour health information and support telephone line. A proportion of HealthLine's callers are referred to the emergency department (ED) for further assessment. The purpose of this study was to gain insight into the appropriateness of these referrals and assess whether they increased the burden on an already strained ED system. METHODS: A list of callers referred from HealthLine to Saskatoon EDs from January 1, 2014, to March 31, 2014 was obtained. This list was cross-referenced with Saskatoon Health Region registration data to determine which of those callers had been registered in one of the three Saskatoon EDs within 48 hours of the original call. RESULTS: During the 90-day time period in question, 707/3,938 (17.9%) of callers were referred by HealthLine to the ED. Out of those referred, 601 were identifiable and 358 attended the ED. Hospital charts were pulled for full data extraction and analysis of the 276 who met inclusion criteria. Of those who presented to the ED and met inclusion criteria, 60% had investigations performed while 66% received some form of treatment. The overall admission rate for the patient population studied was 12.0% v. 16% for non-referred patients. Referred pediatric patients had fewer investigations and treatments with a lower admission rate compared with the adult patients. CONCLUSION: The Saskatchewan HealthLine is doing an effective job at directing callers both to and away from EDs in Saskatoon and not overburdening our local EDs with unnecessary referrals.

Diagnostic accuracy of eFAST in the trauma patient: a systematic review and meta-analysis.

OBJECTIVES: Performing an extended Focused Assessment with Sonography in Trauma (eFAST) exam is common practice in the initial assessment of trauma patients. The objective of this study was to systematically review the published literature on diagnostic accuracy of all components of the eFAST exam. METHODS: We searched Medline and Embase from inception through October 2018, for diagnostic studies examining the sensitivity and specificity of the eFAST exam. After removal of duplicates, 767 records remained for screening, of which 119 underwent full text review. Meta-DiSc™ software was used to create pooled sensitivities and specificities for included studies. Study quality was assessed using the Quality in Prognostic Studies (QUADAS-2) tool. RESULTS: Seventy-five studies representing 24,350 patients satisfied our selection criteria. Studies were published between 1989 and 2017. Pooled sensitivities and specificities were calculated for the detection of pneumothorax (69% and 99% respectively), pericardial effusion (91% and 94% respectively), and intra-abdominal free fluid (74% and 98% respectively). Sub-group analysis was completed for detection of intra-abdominal free fluid in hypotensive (sensitivity 74% and specificity 95%), adult normotensive (sensitivity 76% and specificity 98%) and pediatric patients (sensitivity 71% and specificity 95%). CONCLUSIONS: Our systematic review and meta-analysis suggests that e-FAST is a useful bedside tool for ruling in pneumothorax, pericardial effusion, and intra-abdominal free fluid in the trauma setting. Its usefulness as a rule-out tool is not supported by these results.

OBJECTIF: Le recours à l'évaluation ciblée par échographie étendue en traumatologie (eFAST : sigle anglais) est pratique courante dans l'évaluation initiale des patients ayant subi un trauma. L'étude avait donc pour but une revue systématique de la documentation publiée sur l'exactitude du diagnostic reposant sur tous les éléments constitutifs de l'eFAST. MÉTHODE: Les chercheurs ont effectué une recherche d'études sur les examens de diagnostic ayant pour objets la sensibilité et la spécificité de l'eFAST, dans les bases de données Medline et Embase, depuis leur début respectif jusqu'à octobre 2018. Après le retrait des doubles, il restait 767 documents aux fins de sélection, dont 119 ont été soumis à un examen en texte intégral. Le logiciel Meta-DiScMC a servi à établir la sensibilité et la spécificité globales des études retenues. Quant à la qualité des études, elle a été évaluée à l'aide de l'instrument Quality in Prognostic Studies (QUADAS-2). RÉSULTATS: Au total, 75 études totalisant 24 350 patients et publiées entre 1989 et 2017 répondaient aux critères de sélection. La sensibilité et la spécificité globales ont été calculées pour la détection des pneumothorax (69% et 99% respectivement), des épanchements péricardiques (91% et 94% respectivement) et de liquide libre intra-abdominal (74% et 98% respectivement). Il y a eu également analyse de sous-groupes en vue de la détection de liquide libre intra-abdominal chez les patients hypotendus (sensibilité : 74%; spécificité : 95%), les adultes normotendus (sensibilité : 76%; spécificité : 98%) et les enfants (sensibilité : 71%; spécificité : 95%). CONCLUSION: D'après les résultats de la revue systématique et de la méta-analyse, l'eFAST au chevet se montre utile pour confirmer la présence de pneumothorax, d'épanchement péricardique ou de liquide libre intra-abdominal en traumatologie, mais pas pour en écarter la présence.

Evaluation of emergency department ultrasound machines for the presence of occult blood.

OBJECTIVES: Bedside ultrasound in the emergency department is a common diagnostic tool, especially when evaluating trauma patients. Many trauma patients have blood on their chest and abdomen that may contact the probe during examination. The primary aim of this study was to investigate whether occult blood contamination was present on the emergency department ultrasound machine, both after daily use and after use in trauma. METHODS: For a period of 31 days, the ultrasound machine at the trauma centre emergency department in Saskatoon, Saskatchewan, was tested once daily and following all Level 1 traumas. The ultrasound machine probes and keyboard were swabbed, and contamination was detected using a commercially available phenolphthalein blood testing kit. Any visible blood contamination was also noted. The machine was then cleaned following each positive test and re-tested to ensure the absence of contamination. RESULTS: Over the study period, the ultrasound machine tested positive for occult blood contamination on 10% of daily tests and on 43% of assessments after its use in trauma. The curvilinear probe was most frequently contaminated (daily, 6%; trauma, 26%), followed by the keyboard (daily, 3%; trauma, 26%), but both lacked visible contamination. CONCLUSIONS: In this single centre study, there was evidence of occult blood on the emergency department ultrasound machine after both routine use and major trauma cases, highlighting the need for a standardized cleaning and disinfection protocol.

Computerized physician order entry and decision support improves ED analgesic ordering for renal colic.

OBJECTIVES: Computerized physician order entry (CPOE) offers the potential for safer, faster patient care, as well as greater use of evidence-based therapy via built-in decision support. However, the effectiveness of CPOE in yielding these benefits has shown mixed results in the emergency department (ED) setting. Our objective was to evaluate the impact of CPOE implementation on analgesic prescribing and dosing practices for renal colic presentations. METHODS: This retrospective pre/post comparative study was conducted in 3 tertiary hospitals that implemented CPOE in 2010. Two patient groups were compared: prior to (pre-CPOE) and after (post-CPOE) CPOE implementation. Each group consisted of 230 randomly selected, high-acuity patients presenting to the ED with renal colic. The primary outcome was the proportion of patients receiving ketorolac in the ED. Secondary outcomes included choice of analgesic and average morphine dose. RESULTS: The proportion of patients receiving ketorolac significantly increased after CPOE implementation (65.6% pre-CPOE vs 76.5% post-CPOE, P = .015), as did the proportion of patients receiving fentanyl (pre, 9.7%; post, 16.7%; P = .047). Differences in morphine use (pre, 66.0%; post, 69.1%) and average morphine dose (pre, 10.09 mg; post, 12.28 mg) did not reach statistical significance. CONCLUSIONS: The introduction of CPOE is associated with an increase in ketorolac use for ED renal colic visits. This may reflect the inclusion of ketorolac in the renal colic order set. Computerized physician order entry implementation with condition-specific electronic order sets and decision support may improve evidence-based practice.

Outcomes of children with suspected appendicitis and incompletely visualized appendix on ultrasound.

OBJECTIVES: The objective was to review the clinical outcomes of children with suspected appendicitis after an ultrasound (US) examination fails to fully visualize the appendix, the diagnostic characteristics of US in children with suspected appendicitis, and the predictive value of secondary signs of appendicitis when the appendix is not fully visualized. METHODS: This was a retrospective health record review of children aged 3 to 17 years presenting to a tertiary pediatric emergency department (ED) with suspected appendicitis. Descriptive statistics and diagnostic test characteristics are reported. RESULTS: Overall, 968 children had US. The appendix was fully visualized in 442 cases (45.7%), and 526 (54.3%) children had incompletely visualized appendices. The disposition of those with incompletely visualized appendices were as follows: 59.1% were discharged home, 10.5% went directly to the operating room, and 30.4% were admitted to the hospital for further observation. Of those discharged home based on clinical findings after incompletely visualized appendices, fewer than 0.3% ended up having appendicitis. Ultimately 15.6% of children with incompletely visualized appendices had pathology-confirmed appendicitis. The sensitivity and specificity of US for children with fully visualized appendices were 99.5% (95% confidence interval [CI] = 96.7% to 100%) and 81.3% (95% CI = 75.2% to 86.2%), respectively. The sensitivity and specificity for the presence of any secondary sign in diagnosing appendicitis were 40.2% (95% CI = 29.6% to 51.7%) and 90.6% (95% CI = 87.5% to 93.2%), respectively. CONCLUSIONS: Children with incompletely visualized appendices on US can be safely discharged home based on clinical findings with an acceptable rate of missed appendicitis. Children with nonreassuring clinical examinations following incompletely visualized appendices on US may benefit from further imaging studies prior to appendectomy, to reduce the rate of negative appendectomy. While the presence of secondary signs of inflammation can be used to rule in appendicitis, statistical strength to rule out appendicitis in the absence of secondary signs is insufficient.

A novel role for the SUMO E3 ligase PIAS1 in cancer metastasis.

Tumor metastasis contributes to the grave morbidity and mortality of cancer, but the mechanisms underlying tumor cell invasiveness and metastasis remain incompletely understood. Here, we report that expression of the SUMO E3 ligase PIAS1 suppresses TGFß-induced activation of the matrix metalloproteinase MMP2 in human breast cancer cells. We also find that knockdown of endogenous PIAS1 or inhibition of its SUMO E3 ligase activity stimulates the ability of TGFß to induce an aggressive phenotype in three-dimensional breast cancer cell organoids. Importantly, inhibition of the SUMO E3-ligase activity of PIAS1 in breast cancer cells promotes metastases in mice in vivo. Collectively, our findings define a novel and critical role for the SUMO E3 ligase PIAS1 in the regulation of the invasive and metastatic potential of malignant breast cancer cells. These findings advance our understanding of cancer invasiveness and metastasis with potential implications for the development of biomarkers and therapies in breast cancer.

Identification of a novel function for the chromatin remodeling protein ING2 in muscle differentiation.

The inhibitor of growth (ING) family of zinc-finger plant homeodomain (PHD)-containing chromatin remodeling protein controls gene expression and has been implicated in the regulation of cell proliferation and death. However, the role of ING proteins in cell differentiation remains largely unexplored. Here, we identify an essential function for ING2 in muscle differentiation. We find that knockdown of ING2 by RNA interference (RNAi) blocks the differentiation of C2C12 cells into myotubes, suggesting that ING2 regulates the myogenic differentiation program. We also characterize a mechanism by which ING2 drives muscle differentiation. In structure-function analyses, we find that the leucine zipper motif of ING2 contributes to ING2-dependent muscle differentiation. By contrast, the PHD domain, which recognizes the histone H3K4me3 epigenetic mark, inhibits the ability of ING2 to induce muscle differentiation. We also find that the Sin3A-HDAC1 chromatin remodeling complex, which interacts with ING2, plays a critical role in ING2-dependent muscle differentiation. These findings define a novel function for ING2 in muscle differentiation and bear significant implications for our understanding of the role of the ING protein family in cell differentiation and tumor suppression.

An isoform-specific SnoN1-FOXO1 repressor complex controls neuronal morphogenesis and positioning in the mammalian brain.

Control of neuronal positioning is fundamental to normal brain development. However, the cell-intrinsic mechanisms that govern neuronal positioning remain to be elucidated. Here, we report that the spliced protein products of the transcriptional regulator SnoN, SnoN1 and SnoN2, harbor opposing functions in the coordinate regulation of neuronal branching and positioning. Knockdown of SnoN2 stimulates axon branching in primary neurons and impairs migration of granule neurons in the rat cerebellar cortex in vivo. By contrast, SnoN1 knockdown suppresses SnoN2 knockdown-induced neuronal branching and strikingly triggers excessive migration of granule neurons in the cerebellar cortex. We also find that SnoN1 forms a complex with the transcription factor FOXO1 that represses the X-linked lissencephaly gene encoding doublecortin (DCX). Accordingly, repression of DCX mediates the ability of SnoN1 to regulate branching in primary neurons and granule neuron migration in vivo. These data define an isoform-specific SnoN1-FOXO1 transcriptional complex that orchestrates neuronal branching and positioning in the brain with important implications for the study of developmental disorders of cognition and epilepsy.

Suppression of TGFß-induced epithelial-mesenchymal transition like phenotype by a PIAS1 regulated sumoylation pathway in NMuMG epithelial cells.

Epithelial-mesenchymal-transition (EMT) is a fundamental cellular process that is critical for normal development and tumor metastasis. The transforming growth factor beta (TGFß) is a potent inducer of EMT like effects, but the mechanisms that regulate TGFß-induced EMT remain incompletely understood. Using the widely employed NMuMG mammary epithelial cells as a model to study TGFß-induced EMT, we report that TGFß downregulates the levels of the SUMO E3 ligase PIAS1 in cells undergoing EMT. Gain and loss of function analyses indicate that PIAS1 acts in a SUMO ligase dependent manner to suppress the ability of TGFß to induce EMT in these cells. We also find that TGFß inhibits sumoylation of the PIAS1 substrate SnoN, a transcriptional regulator that antagonizes TGFß-induced EMT. Accordingly, loss of function mutations of SnoN sumoylation impair the ability of SnoN to inhibit TGFß-induced EMT in NMuMG cells. Collectively, our findings suggest that PIAS1 is a novel negative regulator of EMT and reveal that inhibition of the PIAS1-SnoN sumoylation pathway represents a key mechanism by which TGFß induces EMT, with important implications in normal development and tumor metastasis.

Cyclic AMP phosphodiesterase 4D (PDE4D) Tethers EPAC1 in a vascular endothelial cadherin (VE-Cad)-based signaling complex and controls cAMP-mediated vascular permeability.

Vascular endothelial cell (VEC) permeability is largely dependent on the integrity of vascular endothelial cadherin (VE-cadherin or VE-Cad)-based intercellular adhesions. Activators of protein kinase A (PKA) or of exchange protein activated by cAMP (EPAC) reduce VEC permeability largely by stabilizing VE-Cad-based intercellular adhesions. Currently, little is known concerning the nature and composition of the signaling complexes that allow PKA or EPAC to regulate VE-Cad-based structures and through these actions control permeability. Using pharmacological, biochemical, and cell biological approaches we identified and determined the composition and functionality of a signaling complex that coordinates cAMP-mediated control of VE-Cad-based adhesions and VEC permeability. Thus, we report that PKA, EPAC1, and cyclic nucleotide phosphodiesterase 4D (PDE4D) enzymes integrate into VE-Cad-based signaling complexes in human arterial endothelial cells. Importantly, we show that protein-protein interactions between EPAC1 and PDE4D serve to foster their integration into VE-Cad-based complexes and allow robust local regulation of EPAC1-based stabilization of VE-Cad-based adhesions. Of potential translational importance, we mapped the EPAC1 peptide motif involved in binding PDE4D and show that a cell-permeable variant of this peptide antagonizes EPAC1-PDE4D binding and directly alters VEC permeability. Collectively, our data indicate that PDE4D regulates both the activity and subcellular localization of EPAC1 and identify a novel mechanism for regulated EPAC1 signaling in these cells.

A SnoN-Ccd1 pathway promotes axonal morphogenesis in the mammalian brain.

The transcriptional corepressor SnoN is a critical regulator of axonal morphogenesis, but how SnoN drives axonal growth is unknown. Here, we report that gene-profiling analyses in cerebellar granule neurons reveal that the large majority of genes altered upon SnoN knockdown are surprisingly downregulated, suggesting that SnoN may activate transcription in neurons. Accordingly, we find that the transcriptional coactivator p300 interacts with SnoN, and p300 plays a critical role in SnoN-induced axon growth. We also identify the gene encoding the signaling scaffold protein Ccd1 as a critical target of SnoN in neurons. Ccd1 localizes to the actin cytoskeleton, is enriched at axon terminals in neurons, and activates the axon growth-promoting kinase JNK (c-Jun N-terminal protein kinase). Knockdown of Ccd1 in neurons reduces axonal length and suppresses the ability of SnoN to promote axonal growth. Importantly, Ccd1 knockdown in rat pups profoundly impairs the formation of granule neuron parallel fiber axons in the rat cerebellar cortex in vivo. These findings define a novel SnoN-Ccd1 link that promotes axonal growth in the mammalian brain, with important implications for axonal development and regeneration.

ING2 as a novel mediator of transforming growth factor-beta-dependent responses in epithelial cells.

Members of the ING (inhibitor of growth) family of chromatin modifying proteins (ING1-ING5) have emerged as critical regulators of gene expression and cellular responses, suggesting that the ING proteins may impinge on specific signal transduction pathways and their mediated effects. Here, we demonstrate a role for the protein ING2 in mediating responses by the transforming growth factor (TGF)-beta-Smad signaling pathway. We show that ING2 promotes TGF-beta-induced transcription. Both gain-of-function and RNA interference-mediated knockdown of endogenous ING2 reveal that ING2 couples TGF-beta signals to the induction of transcription and cell cycle arrest. We also find that the Smad-interacting transcriptional modulator SnoN interacts with ING2 and promotes the assembly of a protein complex containing SnoN, ING2, and Smad2. Knockdown of endogenous SnoN blocks the ability of ING2 to promote TGF-beta-dependent transcription, and conversely expression of SnoN augments ING2 enhancement of the TGF-beta response. Collectively, our data suggest that ING2 collaborates with SnoN to mediate TGF-beta-induced Smad-dependent transcription and cellular responses.

Both protein kinase A and exchange protein activated by cAMP coordinate adhesion of human vascular endothelial cells.

cAMP regulates integrin-dependent adhesions of vascular endothelial cells (VECs) to extracellular matrix proteins, their vascular endothelial cadherin-dependent intercellular adhesions, and their proliferation and migration in response to growth and chemotactic factors. Previously, we reported that cAMP-elevating agents differentially inhibited migration of human VECs isolated from large vascular structures (macro-VECs, human aortic endothelial cells [HAECs]) or small vascular structures (micro-VECs, human microvascular endothelial cells [HMVECs]) and that cAMP hydrolysis by phosphodiesterase (PDE)3 and PDE4 enzymes was important in coordinating this difference. Here we report that 2 cAMP-effector enzymes, namely protein kinase (PK)A and exchange protein activated by cAMP (EPAC), each regulate extracellular matrix protein-based adhesions of both macro- and micro-VECs. Of interest and potential therapeutic importance, we report that although specific pharmacological activation of EPAC markedly stimulated adhesion of micro-VECs to extracellular matrix proteins when PKA was inhibited, this treatment only modestly promoted adhesion of macro-VECs. Consistent with an important role for cAMP PDEs in this difference, PDE3 or PDE4 inhibitors promoted EPAC-dependent adhesions in micro-VECs when PKA was inhibited but not in macro-VECs. At a molecular level, we identify multiple, nonoverlapping, PKA- or EPAC-based signaling protein complexes in both macro- and micro-VECs and demonstrate that each of these complexes contains either PDE3B or PDE4D but not both of these PDEs. Taken together, our data support the concept that adhesion of macro- and micro-VECs is differentially regulated by cAMP and that these differences are coordinated through selective actions of cAMP at multiple nonoverlapping signaling complexes that contain PKA or EPAC and distinct PDE variants.

Adiponectin, ghrelin, and leptin differentially influence human platelet and human vascular endothelial cell functions: implication in obesity-associated cardiovascular diseases.

A very strong epidemiological link exists between obesity, the metabolic syndrome, diabetes and diabetes-associated cardiovascular pathologies. For this reason the peripheral effects of the centrally-acting satiety adipokines, adiponectin and leptin, and of non-adipose-derived hormones with similar effects, like ghrelin, have received considerable attention. In this report, we have extended our previous studies of the pro-thrombotic effects of leptin and determined the effects of adiponectin or ghrelin on human platelet activation. Thus, while leptin stimulated human platelet aggregation and adhesion, addition of adiponectin or of ghrelin did not affect either aggregation or adhesion of these cells; even at supra-physiological concentrations. In addition, we compared the impact of these three important hormones on microvascular endothelial cell permeability, an important parameter of endothelial function that when impaired contributes to several vascular pathologies. While physiologically relevant concentrations of either leptin or adiponectin increased the integrity of the diffusion barrier formed by a monolayer of human microvascular endothelial cells, only supra-physiological concentrations of ghrelin had this effect. None of these agents reduced microvascular endothelial barrier function. Taken together, our data are consistent with the ideas that leptin activates human platelets and limits transendothelial cell diffusion but that adiponectin only influences endothelial cell permeability. In contrast, ghrelin had neither of these effects. We propose that these data identify important differences in the effects of leptin, adiponectin or ghrelin on microvascular endothelial cells and platelets and may provide a basis on which to pharmacologically manipulate the selective effects of these peptides on these cell types in human cardiovascular or thrombotic diseases associated with obesity.

Sumoylated SnoN represses transcription in a promoter-specific manner.

The transcriptional modulator SnoN controls a diverse set of biological processes, including cell proliferation and differentiation. The mechanisms by which SnoN regulates these processes remain incompletely understood. Recent studies have shown that SnoN exerts positive or negative regulatory effects on transcription. Because post-translational modification of proteins by small ubiquitin-like modifier (SUMO) represents an important mechanism in the control of the activity of transcriptional regulators, we asked if this modification regulates SnoN function. Here, we show that SnoN is sumoylated. Our data demonstrate that the SUMO-conjugating E2 enzyme Ubc9 is critical for SnoN sumoylation and that the SUMO E3 ligase PIAS1 selectively interacts with and enhances the sumoylation of SnoN. We identify lysine residues 50 and 383 as the SUMO acceptor sites in SnoN. Analyses of SUMO "loss-of-function" and "gain-of-function" SnoN mutants in transcriptional reporter assays reveal that sumoylation of SnoN contributes to the ability of SnoN to repress gene expression in a promoter-specific manner. Although this modification has little effect on SnoN repression of the plasminogen activator inhibitor-1 promoter and only modestly potentiates SnoN repression of the p21 promoter, SnoN sumoylation robustly augments the ability of SnoN to suppress transcription of the myogenesis master regulatory gene myogenin. In addition, we show that the SnoN SUMO E3 ligase, PIAS1, at its endogenous levels, suppresses myogenin transcription. Collectively, our findings suggest that SnoN is directly regulated by sumoylation leading to the enhancement of the ability of SnoN to repress transcription in a promoter-specific manner. Our study also points to a physiological role for SnoN sumoylation in the control of myogenin expression in differentiating muscle cells.

Vascular endothelial cell cyclic nucleotide phosphodiesterases and regulated cell migration: implications in angiogenesis.

Angiogenesis is necessary during embryonic development and wound healing but can be detrimental in pathologies, including cancer. Because initiation of angiogenesis involves migration and proliferation of vascular endothelial cells (VECs) and cAMP-elevating agents inhibit these events, such agents may represent a novel therapeutic avenue to controlling angiogenesis. Intracellular cAMP levels are regulated by their synthesis by adenylyl cyclases and hydrolysis by cyclic nucleotide phosphodiesterases (PDEs). In this report, we show that human VECs express variants of PDE2, PDE3, PDE4, and PDE5 families and demonstrate that the levels of these enzymes differ in VECs derived from aorta, umbilical vein, and microvascular structures. Selective inhibition of PDE2 did not increase cAMP in any VECs, whether in the absence or presence of forskolin, but it did inhibit migration of all VECs studied. Inhibition of PDE4 activity decreased migration, and in conjunction with forskolin, increased cAMP in all VECs studied. PDE3 inhibition potentiated forskolin-induced increases in cAMP and inhibited migration in VECs derived from aorta and umbilical vein but not in microvascular VECs. In experiments with combinations of PDE2, PDE3, and PDE4 inhibitors, a complex interaction between the abilities of these agents to limit human VEC migration was observed. Overall, our data are consistent with the hypothesis that PDE subtype inhibition allows different effects in distinct VEC populations and indicate that these agents may represent novel therapeutic agents to limit angiogenesis in complex human diseases.