Association Between Hospital Postoperative Troponin Use and Patient Outcomes After Vascular Surgery.

BACKGROUND: Acute myocardial injury after noncardiac surgery, which is most often symptomatically silent, is associated with increased mortality and morbidity. However, it is not known if routine postoperative troponin testing will affect patient outcomes. METHODS: We assembled a cohort of patients who underwent carotid endarterectomy or abdominal aortic aneurysm repair in Ontario, Canada, from 2010 to 2017. Hospitals were categorized into high, medium, and low troponin testing intensity based on the proportion of patients who received postoperative troponin testing. Cox proportional hazards modeling was used to assess the association between hospital-specific testing intensity and 30-day and 1-year major adverse cardiovascular events (MACEs) while adjusting for patient-, surgery-, and hospital-level factors. RESULTS: The cohort consisted of 18,467 patients from 17 hospitals. Mean age was 72 years, and 74.0% were men. Rates of postoperative troponin testing were 77.5%, 35.8%, and 21.6% in the high-, medium-, and low-testing intensity hospitals, respectively. At 30 days, 5.3%, 5.3%, and 6.5% of patients in high-, medium-, and low-testing intensity hospitals experienced MACE, respectively. Higher troponin testing rate was associated with lower adjusted hazard ratios (HRs) for MACE at 30 days (0.94; 95% confidence interval [CI], 0.89-0.98) and at 1 year (0.97; 95% CI, 0.94-0.99) for each 10% increase in hospital troponin rate. Hospitals with high-testing intensity had higher rates of postoperative cardiology referrals, cardiovascular testing, and rates of new cardiovascular prescriptions. CONCLUSIONS: Patients undergoing vascular surgery at hospitals with higher postoperative troponin testing intensity experienced fewer adverse outcomes than patients who had surgery at hospitals with lower testing intensity.

Adverse Fetal Outcomes and Maternal Mortality Following Nonobstetric Abdominopelvic Surgery in Pregnancy: A Systematic Review and Meta-analysis.

OBJECTIVE: To quantify the absolute risks of adverse fetal outcomes and maternal mortality following nonobstetric abdominopelvic surgery in pregnancy. SUMMARY BACKGROUND DATA: Surgery is often necessary in pregnancy, but absolute measures of risk required to guide perioperative management are lacking. METHODS: We systematically searched MEDLINE, EMBASE, and EvidenceBased Medicine Reviews from January 1, 2000, to December 9, 2020, for observational studies and randomized trials of pregnant patients undergoing nonobstetric abdominopelvic surgery. We determined the pooled proportions of fetal loss, preterm birth, and maternal mortality using a generalized linear random/mixed effects model with a logit link. RESULTS: We identified 114 observational studies (52 [46%] appendectomy, 34 [30%] adnexal, 8 [7%] cholecystectomy, 20 [17%] mixed types) reporting on 67,111 pregnant patients. Overall pooled proportions of fetal loss, preterm birth, and maternal mortality were 2.8% (95% CI 2.2-3.6), 9.7% (95% CI 8.3-11.4), and 0.04% (95% CI 0.02-0.09; 4/10,000), respectively. Rates of fetal loss and preterm birth were higher for pelvic inflammatory conditions (eg, appendectomy, adnexal torsion) than for abdominal or nonurgent conditions (eg, cholecystectomy, adnexal mass). Surgery in the second and third trimesters was associated with lower rates of fetal loss (0.1%) and higher rates of preterm birth (13.5%) than surgery in the first and second trimesters (fetal loss 2.9%, preterm birth 5.6%). CONCLUSIONS: Absolute risks of adverse fetal outcomes after nonobstetric abdom- inopelvic surgery vary with gestational age, indication, and acuity. Pooled estimates derived here identify high-risk clinical scenarios, and can inform implementation of mitigation strategies and improve preoperative counselling.

Troponin testing after noncardiac surgery: a population-based historical cohort study on variation and factors associated with testing in Ontario.

BACKGROUND: International practice guidelines make different recommendations for postoperative troponin testing to detect perioperative myocardial infarction and myocardial injury after noncardiac surgery. To gain insights into current testing patterns, we evaluated predictors of routine troponin testing after three commonly performed major noncardiac surgeries. METHODS: We conducted a population-based historical cohort study of adults having major orthopedic, colorectal, or vascular surgery in Ontario, Canada from 1 January 2010 to 31 December 2017. We used hierarchical logistic regression modelling to assess the association of patient, surgery, and hospital factors with postoperative troponin testing, while accounting for clustering at the hospital level. We characterized hospital-level variation by the intraclass correlation coefficient (ICC), which was adjusted for various characteristics. RESULTS: The cohort included 176,454 eligible patients. Hospital-specific adjusted testing rates ranged from 0-20.1% for orthopedic surgery, 0-43.8% for colorectal surgery, and 19.6-88.0% for vascular surgery. Older age, urgent surgery status, and surgery duration were consistently associated with higher rates of testing for all three surgeries. Higher Revised Cardiac Risk Index scores were associated with higher odds of testing for orthopedic and colorectal surgery, but not for vascular surgery. Even after adjustment, the ICCs were 9.2%, 7.4%, and 24.1% for orthopedic, general, and vascular surgery, respectively. CONCLUSIONS: Troponin testing varied substantially across hospitals for selected major noncardiac surgery procedures even after accounting for differences in patient-level cardiac risk factors. Our observations lend support to a more standardized approach for troponin testing after noncardiac surgery.

RéSUMé: CONTEXTE: Les directives de pratique internationales émettent différentes recommandations en ce qui concerne les dosages postopératoires de troponines afin de détecter l'infarctus du myocarde et les lésions myocardiques périopératoires après une chirurgie non cardiaque. Pour mieux comprendre les habitudes de test actuelles, nous avons évalué les prédicteurs de dosage de troponines de routine après trois chirurgies non cardiaques majeures couramment réalisées. MéTHODE: Nous avons réalisé une étude de cohorte historique basée sur la population d'adultes bénéficiant d'une chirurgie orthopédique, colorectale ou vasculaire majeure en Ontario, au Canada, entre le 1er janvier 2010 et le 31 décembre 2017. Nous avons utilisé un modèle de régression logistique hiérarchique afin d'évaluer l'association des facteurs liés au patient, à la chirurgie et à l'hôpital avec les dosages de troponines postopératoires, tout en tenant compte des groupements au niveau hospitalier. Nous avons caractérisé la variation hospitalière par le coefficient de corrélation intraclasse (CCI), qui a été ajusté pour tenir compte de diverses caractéristiques. RéSULTATS: La cohorte comprenait 176 454 patients éligibles. Les taux de tests ajustés propres à l'hôpital variaient de 0 à 20,1 % pour les chirurgies orthopédiques, de 0 à 43,8 % pour les chirurgies colorectales et de 19,6 à 88,0 % pour les chirurgies vasculaires. Un âge plus avancé, un statut de chirurgie urgente et la durée de la chirurgie étaient systématiquement associés à des taux plus élevés de dosages pour les trois chirurgies. Des scores plus élevés sur l'Indice de risque cardiaque révisé étaient associés à des probabilités plus élevées de dosages pour les chirurgies orthopédiques et colorectales, mais pas pour les chirurgies vasculaires. Même après ajustement, les CCI étaient de 9,2 %, 7,4 % et 24,1 % pour les chirurgies orthopédiques, générales et vasculaires, respectivement. CONCLUSION: Les dosages de troponines varient considérablement d'un hôpital à l'autre pour certaines interventions chirurgicales non cardiaques majeures, même après avoir pris en compte les différences dans les facteurs de risque cardiaques liés au patient. Nos observations appuient une approche plus standardisée des dosages de troponines après une chirurgie non cardiaque.

Renin-angiotensin-aldosterone system inhibitors and major cardiovascular events and acute kidney injury in patients with coronary artery disease.

BACKGROUND: Renin-angiotensin-aldosterone system inhibitors (RAASIs) are recommended for most patients with coronary artery disease (CAD). However, there is debate across guidelines as to which patients with CAD benefit the most from these agents. This study investigated the association between RAASIs and cardiovascular outcomes and acute kidney injury in a contemporary cohort of patients with CAD. METHODS: Patients ≥65 years of age with CAD alive on April 1, 2012 in Ontario, Canada were included. Outcomes included major adverse cardiovascular events (MACE: cardiovascular death, myocardial infarction (MI), unstable angina, stroke, or coronary revascularization), and acute kidney injury (AKI) hospitalizations at 4 years. Inverse probability of treatment-weighted Cox proportional hazards regression models was used to compare the rates of each outcome in patients treated with and without RAASIs (angiotensin-converting enzyme inhibitors or angiotensin II receptor blockers). RESULTS: There were 165,058 patients with CAD identified (mean age 75 years, 65.5% male, 64.7% prescribed RAASIs). After inverse-probability weighting, treatment with RAASIs was associated with a lower rate of MACE compared with treatment without RAASIs (17.6% vs 18.2%, hazard ratio [HR]: 0.96, 95% CI: 0.93-0.99, respectively). However, treatment with RAASIs was associated with a higher rate of AKI compared with treatment without RAASIs (1.7% vs 1.5%, HR: 1.14, 95% CI: 1.02-1.29, respectively). The reduction in MACE was greater in patients with prior MI (HR: 0.87, 95% CI: 0.82-0.92) compared with patients without prior MI (HR: 1.00, 95% CI: 0.97-1.04, interaction p < 0.01). The increase in AKI was lower in patients with prior MI (HR: 0.82, 95% CI: 0.66-1.00) compared with patients without prior MI (HR: 1.37, 95% CI: 1.19-1.57, interaction p < 0.01). CONCLUSIONS: This study supports the continued use of RAASIs in patients with CAD, although the benefit appears smaller in magnitude than observed in prior trials. High-risk patients, particularly those with prior MI, appear to benefit the most from RAASIs.

Troponin Testing After Noncardiac Surgery in Ontario: An Observational Study.

BACKGROUND: In 2017, the Canadian Cardiovascular Society (CCS) published guidelines recommending postoperative troponin surveillance in higher-risk patients having major noncardiac surgery. The objective of this study was to evaluate the proportion of major noncardiac surgery patients that would meet recommendations for troponin testing and to assess the rates of troponin testing before guideline adoption. METHODS: We conducted a retrospective observational study of patients age 40 to 105 undergoing a subset of major noncardiac surgeries that included orthopedics, gynecology, general, urology, vascular, and thoracic surgeries in Ontario, Canada from January 1, 2010 to December 31, 2017. The primary outcomes were the proportion of patients recommended for testing based on the guidelines and rates of troponin testing within 2 days of surgery. RESULTS: We identified 257,704 patients who underwent noncardiac surgery. Mean age was 66.4 ± 11.9 years, and 12.4% underwent urgent surgery. Applying the CCS guidelines, 71.2% of elective surgery patients and 81.0% of urgent surgery patients would have met recommendations for postoperative troponin screening, whereas 10.8% and 27.1% received postoperative troponin testing, respectively. Most elective surgery patients met recommendations for testing based on the age criterion (54.9%), followed by diabetes (24.6%) and high-risk surgery (22.7%) criteria. Troponin testing varied substantially by types of surgery: highest for open abdominal aortic aneurisms and lowest for hysterectomies. CONCLUSIONS: Based on the CCS guidelines, most patients undergoing the subset of surgeries assessed would have met recommendations for routine troponin testing. In contrast, routine troponin testing before guideline adoption was done infrequently in Ontario, with substantial variations based on the surgery type.

INTRODUCTION: En 2017, la Société canadienne de cardiologie (SCC) a publié des lignes directrices dont les recommandations portaient sur la surveillance de la troponine en phase postopératoire chez les patients exposés à un risque accru de subir une intervention chirurgicale non cardiaque importante. L'objectif de la présente étude était d'évaluer le nombre de patients subissant une intervention chirurgicale non cardiaque importante qui répondraient aux recommandations sur le dosage de la troponine et de déterminer la fréquence des dosages de la troponine avant l'adoption des lignes directrices. MÉTHODES: Nous avons mené une étude observationnelle rétrospective auprès de patients âgés de 40 à 105 ans subissant des interventions chirurgicales non cardiaques importantes, à savoir des interventions de chirurgie orthopédique, de chirurgie gynécologique, de chirurgie générale, de chirurgie urologique, de chirurgie vasculaire et de chirurgie thoracique en Ontario, au Canada, du 1er janvier 2010 au 31 décembre 2017. Les principaux critères d'évaluation étaient le nombre de patients pour qui le dosage était recommandé selon les lignes directrices, et la fréquence des dosages de la troponine dans les deux jours après l'intervention chirurgicale. RÉSULTATS: Nous avons relevé 257 704 patients qui avaient subi une intervention chirurgicale non cardiaque. L'âge moyen était de 66,4 ± 11,9 ans, et 12,4 % avaient subi une intervention chirurgicale urgente. En appliquant les lignes directrices de la SCC, 71,2 % des patients avaient subi une intervention chirurgicale élective et 81,0 % des patients qui avaient subi une intervention chirurgicale urgente répondaient aux recommandations de dépistage de la troponine en phase postopératoire, alors que respectivement 10,8 % et 27,1 % avaient reçu le dosage de la troponine en phase postopératoire. La plupart des patients qui avaient subi une intervention chirurgicale élective répondaient aux recommandations sur le dosage selon le critère d'âge (54,9 %), puis selon le critère de diabète (24,6 %) et le critère d'intervention chirurgicale à risque élevé (22,7 %). Le dosage de la troponine variait de façon substantielle selon le type d'intervention chirurgicale : le dosage le plus élevé lors des traitements chirurgicaux ouverts des anévrismes de l'artère abdominale et le dosage le plus faible lors d'hystérectomies. CONCLUSIONS: Selon les lignes directrices de la SCC, la plupart des patients qui subissaient les interventions chirurgicales évaluées avaient répondu aux recommandations de dosage systématique de la troponine. En revanche, le dosage systématique de la troponine avant l'adoption des lignes directrices était rarement réalisé en Ontario, et des variations substantielles selon le type d'intervention chirurgicale étaient observées.

The cost-effectiveness of transcatheter aortic valve replacement in low surgical risk patients with severe aortic stenosis.

AIMS: The economic value of transcatheter aortic valve replacement (TAVR) in low surgical risk patients with severe, symptomatic aortic stenosis is not known. Our objective was to determine the cost-effectiveness of balloon-expandable TAVR and self-expandable TAVR relative to surgical aortic valve replacement (SAVR) in low-risk patients. METHODS AND RESULTS: A fully probabilistic Markov cohort model was constructed to estimate differences in costs and effectiveness [quality-adjusted life years (QALYs)] over the patient's life-time time from the third-party payer's perspective. Clinical outcomes modelled were alive/well (no complications), permanent stroke, ≥moderate paravalvular leak, new pacemaker, rehospitalization, and death. A network meta-analysis of the PARTNER 3 and Evolut Low Risk trial was performed to compare balloon-expandable TAVR, self-expandable TAVR, and SAVR for the efficacy inputs. Incremental-cost effectiveness ratios (ICER) were calculated. The total life-time costs in the balloon-expandable TAVR, self-expandable-TAVR, and SAVR arms were $37 330 ± 4724, $39 660 ± 4862, and $34 583 ± 6731, respectively, and total life-time QALYs gained were 9.15 ± 3.23, 9.13 ± 3.23, and 9.05 ± 3.20, respectively. The ICERs for balloon-expandable TAVR and self-expandable TAVR against SAVR were $27 196/QALY and $59 641/QALY, respectively. Balloon-expandable TAVR was less costly and more effective than self-expandable TAVR. There was substantial uncertainty, with 53% and 58% of model iterations showing balloon-expandable TAVR to be the preferred option at willingness-to-pay thresholds of $50 000/QALY and $100 000/QALY, respectively. CONCLUSION: Compared with SAVR, TAVR, particularly with balloon-expandable prostheses may be a cost-effective option for patients with severe aortic stenosis at low surgical risk.

Calibration and discrimination of the Framingham Risk Score and the Pooled Cohort Equations.

BACKGROUND: Although accurate risk prediction is essential in guiding treatment decisions in primary prevention of atherosclerotic cardiovascular disease, the accuracy of the Framingham Risk Score (recommended by a Canadian guideline) and the Pooled Cohort Equations (recommended by US guidelines) has not been assessed in a large contemporary Canadian population. Our primary objective was to assess the calibration and discrimination of the Framingham Risk Score and Pooled Cohort Equations in Ontario, Canada. METHODS: We conducted an observational study involving Ontario residents aged 40 to 79 years, without a history of atherosclerotic cardiovascular disease, who underwent cholesterol testing and blood pressure measurement from Jan. 1, 2010, to Dec. 31, 2014. We compared predicted event rates generated by the Framingham Risk Score and the Pooled Cohort Equations with observed event rates at 5 years using linkages from validated administrative databases. RESULTS: Our study cohort included 84 617 individuals (mean age 56.3 yr, 56.9% female). Over a maximum follow-up period of 5 years, we observed 2162 (2.6%) events according to the outcome definition of the Framingham Risk Score, and 1224 (1.4%) events according to the outcome definition of the Pooled Cohort Equations. The predicted event rate of 5.78% by the Framingham Risk Score and 3.51% by the Pooled Cohort Equations at 5 years overestimated observed event rates by 101% and 115%, respectively. The degree of overestimation differed by age and ethnicity. The C statistics for the Framingham Risk Score (0.74) and Pooled Cohort Equations (0.73) were similar. INTERPRETATION: The Framingham Risk Score and Pooled Cohort Equations significantly overpredicted the actual risks of atherosclerotic cardiovascular disease events in a large population from Ontario. Our finding suggests the need for further refinement of cardiovascular disease risk prediction scores to suit the characteristics of a multiethnic Canadian population.

Clinical Outcomes With Beta-Blocker Use in Patients With Recent History of Myocardial Infarction.

BACKGROUND: It is uncertain whether beta-blockers (BBs) are beneficial in contemporary stable patients with prior myocardial infarction (MI). Therefore, we sought to examine the effectiveness of BB use in this population. METHODS: We conducted a cohort study with the use of administrative databases of patients ≥ 65 years of age, alive on April 1, 2012 (index date) with a hospital discharge diagnosis of MI within the previous 3 years. The primary outcome was time to death or hospitalization for MI or angina 1 year after the index date, with inverse probability of treatment weighting. RESULTS: We included 33,811 patients with prior MI, of whom 21,440 (63.4%) were dispensed a BB. The median age was 78 years, and 56% were male. There was no difference in the 1-year hazard of death/hospitalization for MI or angina (14.8% vs 14.7%, hazard ratio 1.00, 95% confidence interval 0.94-1.07; P = 0.90) in those receiving vs not receiving BB. Similarly, there was no difference in the individual end points in composite nor in 3-year outcomes. Subgroup analysis by age, sex, MI timing, MI type, heart failure, and atrial fibrillation found no benefit. Patients with a history of revascularisation treated with BBs had a lower rate of the composite outcome compared with those without such history (P = 0.006 for interaction) at 1 year but not at 3 years. CONCLUSIONS: In this large contemporary population-based observational study of older stable patients with prior MI, BBs were not associated with a reduction in major cardiovascular events or mortality in those with MI within the previous 3 years. This study supports the need to conduct contemporary clinical trials evaluating the use of BBs after MI.

Evaluation of Machine Learning Algorithms for Predicting Readmission After Acute Myocardial Infarction Using Routinely Collected Clinical Data.

BACKGROUND: The ability to predict readmission accurately after hospitalization for acute myocardial infarction (AMI) is limited in current statistical models. Machine-learning (ML) methods have shown improved predictive ability in various clinical contexts, but their utility in predicting readmission after hospitalization for AMI is unknown. METHODS: Using detailed clinical information collected from patients hospitalized with AMI, we evaluated 6 ML algorithms (logistic regression, naïve Bayes, support vector machines, random forest, gradient boosting, and deep neural networks) to predict readmission within 30 days and 1 year of discharge. A nested cross-validation approach was used to develop and test models. We used C-statistics to compare discriminatory capacity, whereas the Brier score was used to indicate overall model performance. Model calibration was assessed using calibration plots. RESULTS: The 30-day readmission rate was 16.3%, whereas the 1-year readmission rate was 45.1%. For 30-day readmission, the discriminative ability for the ML models was modest (C-statistic 0.641; 95% confidence interval (CI), 0.621-0.662 for gradient boosting) and did not outperform previously reported methods. For 1-year readmission, different ML models showed moderate performance, with C-statistics around 0.72. Despite modest discriminatory capabilities, the observed readmission rates were markedly higher in the tenth decile of predicted risk compared with the first decile of predicted risk for both 30-day and 1-year readmission. CONCLUSIONS: Despite including detailed clinical information and evaluating various ML methods, these models did not have better discriminatory ability to predict readmission outcomes compared with previously reported methods.

Comparative effectiveness of ACE inhibitors and angiotensin receptor blockers in patients with prior myocardial infarction.

Objective: Although ACE inhibitors (ACEIs) and angiotensin receptor blockers (ARBs) are commonly prescribed for patients with coronary artery disease, whether these medications are similarly effective is still a subject of intense debate. Our objective was to compare the clinical effectiveness of ACEIs and ARBs in patients with prior myocardial infarction (MI). Methods: All residents older than 65 years, alive on 1 April 2012, with a prior MI were included. Propensity weighting was used to balance potentially confounding baseline covariates between the treatment groups. The primary outcome was a composite of cardiovascular death, hospitalisation for MI or unstable angina at 3 years. Results: Our cohort included 59 353 patients with MI; their mean age was 77 years and 40% were women. In the propensity-weighted cohort, the primary outcome occurred in 6.5% in the ACEI group and 5.7% in the ARB group at 1 year (HR comparing ACEI with ARB 1.14, 95% CI 1.05 to 1.23, p<0.001). At 3 years, the primary outcome occurring in 16.0% with ACEIs and 15.1% with ARBs (HR 1.07; 95% CI 1.02 to 1.12; p<0.001). A significant interaction with sex was observed, with women prescribed ACEIs having a higher hazards (HR 1.17; 95% CI 1.10 to 1.26) compared with ARBs, while no significant difference was seen among men (HR 1.00; 95% CI 0.93 to 1.06, interaction p<0.001). Conclusions: Despite previous concerns regarding ARBs, we found that they had slightly lower rates of adverse clinical cardiovascular outcomes among older patients with MI compared with ACEIs. The observed difference in clinical outcomes may be related to a sex difference in effectiveness.

Left ventricular strain analysis using cardiac magnetic resonance imaging in patients undergoing in-centre nocturnal haemodialysis.

AIM: Intensified haemodialysis is associated with regression of left ventricular (LV) mass. Compared to LV ejection fraction, LV strain allows more direct assessment of LV function. We sought to assess the impact of in-centre nocturnal haemodialysis (INHD) on global LV strain (radial, circumferential, and longitudinal) and torsion by cardiac MRI (CMR). METHODS: In this prospective, two-centre cohort study, 37 participants on conventional haemodialysis (CHD, 3-4 h/session for three sessions/week) converted to INHD (7-8 h/session for three sessions/week) and 30 participants continued CHD. Participants underwent CMR using a standardized protocol and had biomarker measurements at baseline and 52 weeks. RESULTS: Among the 55 participants (mean age 55; 40% women) with complete CMR data, those who converted to INHD had a significant improvement in their global circumferential strain (GCS, P = 0.025), while those continuing CHD did not have any significant changes in LV strain. When the two groups were compared, there was significant improvement in torsion. LV strains were significantly correlated with each other, but not with troponin I, C-reactive protein, or brain natriuretic protein (NT-proBNP), except for global longitudinal strain (GLS) with troponin I (P = 0.001) and NT-proBNP (P = 0.038). CONCLUSION: Conversion to INHD was associated with significant improvement in GCS over one year of study, although comparisons with the CHD group were not significant. There was also a significant decrease in torsion in the INHD group compared with CHD. Improvement in LV regional function would support the notion that INHD has favourable effects on both LV structure and function.

Prostaglandin I2 Receptor Agonism Preserves ß-Cell Function and Attenuates Albuminuria Through Nephrin-Dependent Mechanisms.

Discovery of common pathways that mediate both pancreatic ß-cell function and end-organ function offers the opportunity to develop therapies that modulate glucose homeostasis and separately slow the development of diabetes complications. Here, we investigated the in vitro and in vivo effects of pharmacological agonism of the prostaglandin I2 (IP) receptor in pancreatic ß-cells and in glomerular podocytes. The IP receptor agonist MRE-269 increased intracellular 3',5'-cyclic adenosine monophosphate (cAMP), augmented glucose-stimulated insulin secretion (GSIS), and increased viability in MIN6 ß-cells. Its prodrug form, selexipag, augmented GSIS and preserved islet ß-cell mass in diabetic mice. Determining that this preservation of ß-cell function is mediated through cAMP/protein kinase A (PKA)/nephrin-dependent pathways, we found that PKA inhibition, nephrin knockdown, or targeted mutation of phosphorylated nephrin tyrosine residues 1176 and 1193 abrogated the actions of MRE-269 in MIN6 cells. Because nephrin is important to glomerular permselectivity, we next set out to determine whether IP receptor agonism similarly affects nephrin phosphorylation in podocytes. Expression of the IP receptor in podocytes was confirmed in cultured cells by immunoblotting and quantitative real-time PCR and in mouse kidneys by immunogold electron microscopy, and its agonism 1) increased cAMP, 2) activated PKA, 3) phosphorylated nephrin, and 4) attenuated albumin transcytosis. Finally, treatment of diabetic endothelial nitric oxide synthase knockout mice with selexipag augmented renal nephrin phosphorylation and attenuated albuminuria development independently of glucose change. Collectively, these observations describe a pharmacological strategy that posttranslationally modifies nephrin and the effects of this strategy in the pancreas and in the kidney.

A novel assay uncovers an unexpected role for SR-BI in LDL transcytosis.

AIMS: Retention of low-density lipoprotein (LDL) cholesterol beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. Since the overlying endothelium is healthy and intact early on, it is likely that LDL passes through endothelial cells by transcytosis. However, technical challenges have made confirming this notion and elucidating the mechanisms of transcytosis difficult. We developed a novel assay for measuring LDL transcytosis in real time across coronary endothelial cell monolayers; we used this approach to identify the receptor involved. METHODS AND RESULTS: Murine aortas were perfused ex vivo with LDL and dextran of a smaller molecular radius. LDL (but not dextran) accumulated under the endothelium, indicating that LDL transcytosis occurs in intact vessels. We then confirmed that LDL transcytosis occurs in vitro using human coronary artery endothelial cells. An assay was developed to quantify transcytosis of DiI-LDL in real time using total internal reflection fluorescence microscopy. DiI-LDL transcytosis was inhibited by excess unlabelled LDL, while degradation of the LDL receptor by PCSK9 had no effect. Instead, LDL colocalized partially with the scavenger receptor SR-BI and overexpression of SR-BI increased LDL transcytosis; knockdown by siRNA significantly reduced it. Excess HDL, the canonical SR-BI ligand, significantly decreased LDL transcytosis. Aortas from SR-BI-deficient mice were perfused ex vivo with LDL and accumulated significantly less sub-endothelial LDL compared with wild-type littermates. CONCLUSION: We developed an assay to quantify LDL transcytosis across endothelial cells and discovered an unexpected role for SR-BI. Elucidating the mechanisms of LDL transcytosis may identify novel targets for the prevention or therapy of atherosclerosis.

Palmitate-induced inflammatory pathways in human adipose microvascular endothelial cells promote monocyte adhesion and impair insulin transcytosis.

Obesity is associated with inflammation and immune cell recruitment to adipose tissue, muscle and intima of atherosclerotic blood vessels. Obesity and hyperlipidemia are also associated with tissue insulin resistance and can compromise insulin delivery to muscle. The muscle/fat microvascular endothelium mediates insulin delivery and facilitates monocyte transmigration, yet its contribution to the consequences of hyperlipidemia is poorly understood. Using primary endothelial cells from human adipose tissue microvasculature (HAMEC), we investigated the effects of physiological levels of fatty acids on endothelial inflammation and function. Expression of cytokines and adhesion molecules was measured by RT-qPCR. Signaling pathways were evaluated by pharmacological manipulation and immunoblotting. Surface expression of adhesion molecules was determined by immunohistochemistry. THP1 monocyte interaction with HAMEC was measured by cell adhesion and migration across transwells. Insulin transcytosis was measured by total internal reflection fluorescence microscopy. Palmitate, but not palmitoleate, elevated the expression of IL-6, IL-8, TLR2 (Toll-like receptor 2), and intercellular adhesion molecule 1 (ICAM-1). HAMEC had markedly low fatty acid uptake and oxidation, and CD36 inhibition did not reverse the palmitate-induced expression of adhesion molecules, suggesting that inflammation did not arise from palmitate uptake/metabolism. Instead, inhibition of TLR4 to NF-κB signaling blunted palmitate-induced ICAM-1 expression. Importantly, palmitate-induced surface expression of ICAM-1 promoted monocyte binding and transmigration. Conversely, palmitate reduced insulin transcytosis, an effect reversed by TLR4 inhibition. In summary, palmitate activates inflammatory pathways in primary microvascular endothelial cells, impairing insulin transport and increasing monocyte transmigration. This behavior may contribute in vivo to reduced tissue insulin action and enhanced tissue infiltration by immune cells.

Clathrin-dependent entry and vesicle-mediated exocytosis define insulin transcytosis across microvascular endothelial cells.

Transport of insulin across the microvasculature is necessary to reach its target organs (e.g., adipose and muscle tissues) and is rate limiting in insulin action. Morphological evidence suggests that insulin enters endothelial cells of the microvasculature, and studies with large vessel-derived endothelial cells show insulin uptake; however, little is known about the actual transcytosis of insulin and how this occurs in the relevant microvascular endothelial cells. We report an approach to study insulin transcytosis across individual, primary human adipose microvascular endothelial cells (HAMECs), involving insulin uptake followed by vesicle-mediated exocytosis visualized by total internal reflection fluorescence microscopy. In this setting, fluorophore-conjugated insulin exocytosis depended on its initial binding and uptake, which was saturable and much greater than in muscle cells. Unlike its degradation within muscle cells, insulin was stable within HAMECs and escaped lysosomal colocalization. Insulin transcytosis required dynamin but was unaffected by caveolin-1 knockdown or cholesterol depletion. Instead, insulin transcytosis was significantly inhibited by the clathrin-mediated endocytosis inhibitor Pitstop 2 or siRNA-mediated clathrin depletion. Accordingly, insulin internalized for 1 min in HAMECs colocalized with clathrin far more than with caveolin-1. This study constitutes the first evidence of vesicle-mediated insulin transcytosis and highlights that its initial uptake is clathrin dependent and caveolae independent.

AMPK phosphorylation of ACC2 is required for skeletal muscle fatty acid oxidation and insulin sensitivity in mice.

AIMS/HYPOTHESIS: Obesity is characterised by lipid accumulation in skeletal muscle, which increases the risk of developing insulin resistance and type 2 diabetes. AMP-activated protein kinase (AMPK) is a sensor of cellular energy status and is activated in skeletal muscle by exercise, hormones (leptin, adiponectin, IL-6) and pharmacological agents (5-amino-4-imidazolecarboxamide ribonucleoside [AICAR] and metformin). Phosphorylation of acetyl-CoA carboxylase 2 (ACC2) at S221 (S212 in mice) by AMPK reduces ACC activity and malonyl-CoA content but the importance of the AMPK-ACC2-malonyl-CoA pathway in controlling fatty acid metabolism and insulin sensitivity is not understood; therefore, we characterised Acc2 S212A knock-in (ACC2 KI) mice. METHODS: Whole-body and skeletal muscle fatty acid oxidation and insulin sensitivity were assessed in ACC2 KI mice and wild-type littermates. RESULTS: ACC2 KI mice were resistant to increases in skeletal muscle fatty acid oxidation elicited by AICAR. These mice had normal adiposity and liver lipids but elevated contents of triacylglycerol and ceramide in skeletal muscle, which were associated with hyperinsulinaemia, glucose intolerance and skeletal muscle insulin resistance. CONCLUSIONS/INTERPRETATION: These findings indicate that the phosphorylation of ACC2 S212 is required for the maintenance of skeletal muscle lipid and glucose homeostasis.