The Dutch chronic lower limb-threatening ischemia registry (THRILLER): A study protocol for popliteal and infrapopliteal endovascular interventions.

INTRODUCTION: Chronic limb-threatening ischemia (CLTI) is the end stage of peripheral arterial disease (PAD) and is associated with high amputation rates, mortality and disease-related health care costs. In infrapopliteal arterial disease (IPAD), endovascular revascularization should be considered for the majority of anatomical and clinical subgroups of CLTI. However, a gap of high-quality evidence exists in this field. The aim of the Dutch Chronic Lower Limb-Threatening Ischemia Registry (THRILLER) is to collect real world data on popliteal and infrapopliteal endovascular interventions. METHODS: THRILLER is a clinician-driven, prospective, multicenter, observational registry including all consecutive patients that undergo a popliteal or infrapopliteal endovascular intervention in seven Dutch hospitals. We estimate that THRILLER will include 400-500 interventions annually. Standardized follow-up visits with wound monitoring, toe pressure measurement and duplex ultrasonography will be scheduled at 6-8 weeks and 12 months after the intervention. The independent primary endpoints are primary patency, limb salvage and amputation free survival. Patients must give informed consent before participation and will be included according to predefined reporting standards. A data log of patients who meet the inclusion criteria but are not included in the registry will be maintained. We intend to conduct the first interim analysis two years after the start of inclusion. The results will be published in a scientific journal. DISCUSSION: Despite innovations in medical therapy and revascularization techniques, patients with CLTI undergoing endovascular revascularization still have a moderate prognosis. Previous prospective cohort studies were hampered by small sample sizes or heterogeneous reporting. Randomized controlled trials (RCTs) have high costs, potential conflicts of interest and give a limited reflection of daily practice. THRILLER aims to provide the largest prospective well phenotyped up-to-date dataset on treatment outcomes in CLTI patients to answer multiple underexplored research questions regarding diagnostics, medication, patient selection, treatment strategies and post intervention follow-up.

Evaluating the effect of glycation on lipase activity using boronate affinity chromatography and mass spectrometry.

Protein glycation may occur naturally when reducing sugars and proteins coexist, which is often the case for industrial enzymes. The impact of post-translational modifications on enzyme performance (e.g., stability or function) is often not predictable, highlighting the importance of having appropriate analytical methodologies to monitor the influence of glycation on performance. Here, a boronate affinity chromatography method was developed to enrich glycated species followed by mass spectrometry for structural characterization and activity assays for functional assessment. This approach was applied to a (temperature-stressed) lipase used for food applications revealing that storage at -20 °C and 4 °C resulted in minor glycation (below 9%), whereas storage at 25 °C led to a higher glycation level with up to four sugars per lipase molecule. Remarkably, activity measurements revealed that glycation did not reduce lipase activity or stability. Altogether, this novel strategy is a helpful extension to the current analytical toolbox supporting development of enzyme products.

Native Structural and Functional Proteoform Characterization of the Prolyl-Alanyl-Specific Endoprotease EndoPro from Aspergillus niger.

The prolyl-alanyl-specific endoprotease (EndoPro) is an industrial enzyme produced in Aspergillus niger. EndoPro is mainly used for food applications but also as a protease in proteomics. In-depth characterization of this enzyme is essential to understand its structural features and functionality. However, there is a lack of analytical methods capable of maintaining both the structural and functional integrity of separated proteoforms. In this study, we developed an anion exchange (AEX) method coupled to native mass spectrometry (MS) for profiling EndoPro proteoforms. Moreover, we investigated purified EndoPro proteoforms with complementary MS-based approaches, including released N-glycan and glycopeptide analysis, to obtain a comprehensive overview of the structural heterogeneity. We showed that EndoPro has at least three sequence variants and seven N-glycosylation sites occupied by high-mannose glycans that can be phosphorylated. Each glycosylation site showed high microheterogeneity with ∼20 glycans per site. The functional characterization of fractionated proteoforms revealed that EndoPro proteoforms remained active after AEX-separation and the specificity of these proteoforms did not depend on N-glycan phosphorylation. Nevertheless, our data confirmed a strong pH dependence of EndoPro cleavage activity. Altogether, our study demonstrates that AEX-MS is an excellent tool to characterize complex industrial enzymes under native conditions.

Covered stent grafts for relining of chronically occluded femoro-popliteal bypasses in frail patients.

In four high-risk patients with chronically occluded femoro-popliteal bypass suffering from Rutherford 4-5 chronic limb threatening ischemia we performed, as an alternative for redo surgery, endovasculair relining with covered stent grafts. During follow-up (3, 8, 14 and 20 months) one patient had redo percutaneous intervention and eventually below-the-knee amputation.

Experience With the Absorb Everolimus-Eluting Bioresorbable Vascular Scaffold in Arteries Below the Knee: 12-Month Clinical and Imaging Outcomes.

OBJECTIVES: The aim of this study was to investigate the midterm performance of an everolimus-eluting, bioresorbable vascular scaffold (Absorb, Abbott Vascular, Santa Clara, California) for the treatment of focal tibial and distal popliteal lesions. BACKGROUND: Drug-eluting stents are used below the knee to improve technical success and durability, but the ongoing presence of a permanent metal scaffold may have deleterious effects on the local vessel. METHODS: Tibial and distal popliteal angioplasty with scaffold placement was performed using an everolimus-eluting, bioresorbable scaffold (Absorb). Clinical and ultrasound follow-up was performed at 1, 3, 6, 12, and 24 months to detect binary restenosis and evaluate safety, restenosis, and clinical improvement. RESULTS: Thirty-eight limbs in 33 patients were treated for critical limb ischemia (68.4%) or severe claudication (31.6%). Fifty scaffolds were used to treat a total of 43 lesions, with a mean length of 19.2 ± 11.6 mm. During a mean follow-up period of 12.0 ± 3.9 months, 5 patients died, and all others were available for follow-up. Among the 38 treated limbs, clinical improvement was present in 30 (79%). Binary restenosis was detected in 3 of 50 scaffolds (6%). Using the Kaplan-Meier method, rates of primary patency were 96% and 84.6% at 12 and 24 months, respectively, and rates of freedom from clinically driven target lesion revascularization were 96% and 96% at 12 and 24 months, respectively. Complete wound healing occurred in 64% of those treated for tissue loss, with no major amputation and a limb-salvage rate of 100%. CONCLUSIONS: Twelve-month follow-up demonstrated excellent safety, patency, and freedom from target lesion revascularization using the Absorb bioresorbable vascular scaffold below the knee.

A Real-World Experience With the Supera Interwoven Nitinol Stent in Femoropopliteal Arteries: Midterm Patency Results and Failure Analysis.

PURPOSE: To evaluate the safety and midterm patency of the Supera interwoven nitinol stent in a real-world population and determine deployment and patient-related factors that may predispose to loss of patency. METHODS: A retrospective analysis was conducted of 111 consecutive limbs from 97 patients (mean age 75.3 years; 68 men) with severe atherosclerotic disease of the superficial femoral and popliteal arteries that were treated with Supera stents between June 2012 and October 2014. Half the patients had claudication (56%); the remainder had rest pain (19%) and tissue loss (26%). Forty-eight (43%) lesions were chronic total occlusions, and more than half were classified as TransAtlantic Inter-Society Consensus C (22%) or D (30%). RESULTS: All 146 Supera stents (1.32 stents per limb) were deployed successfully, extending over a mean length of 175.5±130.5 mm to treat lesions averaging 151.5±127.1 mm long. At 30 days, Kaplan-Meier estimated freedom from death, target lesion revascularization, and amputation was 97.3%. Primary patency and freedom from clinically driven target lesion revascularization rates were 87.1% and 95.0% at 6 months, respectively, and 78.9% and 87.6% at 12 months, respectively. Four distinct mechanisms for failure were identified in the 13 limbs in which patency was lost; stent intussusception (n=4), compromised inflow or outflow (n=2), gross oversizing (n=1), and neoplastic thrombophilia (n=1); the cause of 5 occlusions could not be identified. CONCLUSION: In this heterogeneous group that included long and complex atheromatous femoropopliteal lesions, the Supera stent achieved excellent clinical and patency results at 1 year. Further improvement may be achieved through careful patient selection and the avoidance of deployment pitfalls.

Properties of Hemoglobin Decolorized with a Histidine-Specific Protease.

This study investigated the application of Aspergilloglutamic peptidase (AGP) on porcine hemoglobin decolorization. AGP from fungus Aspergillus niger is identified to possess a high preference towards the histidine residues. As histidine residues in hemoglobin are known to coordinate the heme group within the globin molecule, we therefore hypothesized that incubating hemoglobin with a histidine-specific protease would efficiently separate the non-heme peptides from the heme-enriched peptides with a minimum degree of hydrolysis. AGP-decolored porcine hemoglobin hydrolysates were assessed on their functional (for example, color, emulsification, foaming, and water binding) and sensory properties. The results were compared with commercially available blood-derived proteins (subtilisin-decolored hemoglobin hydrolysates and plasma protein). It was observed that AGP is able to effectively decolor hemoglobin. The degree of hydrolysis (DH) increased less than 3% using AGP to achieve 90% color reduction of hemoglobin, whereas a DH increase of more than 20% is needed using subtilisin. The AGP-decolored hemoglobin hydrolysates (AGP-Hb) possess good emulsification, foaming, and water binding properties, which are better or comparable with the plasma protein, and much better than the subtilisin-decolored hemoglobin hydrolysates (subtilisin-Hb). The model canned meat with addition of AGP-Hb showed the highest value in hardness, springiness, and chewiness from the texture analysis. Furthermore, the canned meat with AGP-Hb was found to have a better sensory profile than the ones with addition of subtilisin-Hb and plasma protein.

Initial experience with the absorb bioresorbable vascular scaffold below the knee: six-month clinical and imaging outcomes.

PURPOSE: To investigate a new bioresorbable vascular scaffold for the treatment of focal tibial and distal popliteal lesions. METHODS: Tibial and distal popliteal angioplasty was performed in 15 limbs of 14 patients (9 men; median age 82 years) with critical limb ischemia (CLI, n=7) or severe claudication. The 18 lesions (mean length 22.2±14.0 mm) were implanted with 22 everolimus-eluting bioresorbable scaffolds (Absorb). Clinical and ultrasound follow-up was performed at 1, 3, 6, and 12 months to detect restenosis and evaluate safety, midterm restenosis rate, and clinical improvement. RESULTS: Immediate technical success was 100%, although a single limb suffered 2 scaffold thromboses on the first day; it was salvaged with repeat endovascular intervention. All patients were available for surveillance examinations during a follow-up of 6.1±3.9 months; no patient died. Of the 15 limbs in the analysis, clinical improvement was present in 12 (4 of 7 CLI patients); there was no amputation, bypass surgery, or evidence of binary restenosis on follow-up sonographic examination. CONCLUSION: Midterm follow-up for this small pilot sample demonstrates acceptable safety and patency results, together with freedom from all major adverse limb events, using the Absorb bioresorbable vascular scaffold below the knee.

A non-canonical NRPS is involved in the synthesis of fungisporin and related hydrophobic cyclic tetrapeptides in Penicillium chrysogenum.

The filamentous fungus Penicillium chrysogenum harbors an astonishing variety of nonribosomal peptide synthetase genes, which encode proteins known to produce complex bioactive metabolites from simple building blocks. Here we report a novel non-canonical tetra-modular nonribosomal peptide synthetase (NRPS) with microheterogenicity of all involved adenylation domains towards their respective substrates. By deleting the putative gene in combination with comparative metabolite profiling various unique cyclic and derived linear tetrapeptides were identified which were associated with this NRPS, including fungisporin. In combination with substrate predictions for each module, we propose a mechanism for a 'trans-acting' adenylation domain.

Non-coronary atherosclerosis.

During the last decades, the clinical and research interest in atherosclerosis has been mostly focused on coronary arteries. After the publications of the European Society Guidelines and AHA/ACC Guidelines on Peripheral artery diseases, and of the Registry REduction in Atherothrombosis for Continued Health Registry, there has been an increased interest in atherosclerosis of the lower extremity arteries and its presence in multifocal disease. However, awareness in the general population and the medical community of non-coronary artery diseases, and of its major prognostic implications remain relatively low. The aim of this general review stemming out of an ESC Working Group on Peripheral Circulation meeting in 2011 is to enhance awareness of this complex disease highlighting the importance of the involvement of atherosclerosis at different levels with respect to clinical presentation, diagnosis, and co-existence of the disease in the distinct arterial territories. We also emphasize the need of an interdisciplinary approach to face the broad and complex spectrum of multifocal disease, and try to propose a series of tentative recommendations and measures to be implemented in non-coronary atherosclerosis.

Sudden death during follow-up after new-onset ventricular tachycardia in vascular surgery patients.

BACKGROUND: Vascular surgery patients are at increased risk for late sudden cardiac death. Identification of patients at risk during surgery offers the opportunity for focused therapy. METHODS: We monitored 483 vascular surgery patients who had no documented history of arrhythmias to identify perioperative new-onset ventricular tachyarrhythmia (VT) and myocardial ischemia using a continuous electrocardiographic (ECG) device for 72 hours. Cardiac risk factors, left ventricular ejection fraction (LVEF), medical therapy, inflammation status, and perioperative ischemia in relation to arrhythmia were noted in all patients. During follow-up, event-based outcomes analysis was used to describe survival. RESULTS: New-onset perioperative VT was detected in 33 patients (6.8%). A higher percentage of patients experiencing perioperative VT had reduced LVEF preoperatively than those without VT (24% vs 12%; P = .04). Additionally, fewer patients experiencing VT were receiving statins than those without (70% vs 85%; P = .02). Patients experiencing VT had a higher incidence of myocardial ischemia (30% vs 18%; P = .10). Perioperative VT was preceded by ischemia in only 60% of the cases. The overall cohort survival was 83% at 24-month follow-up (interquartile range [IQR], 1.1-1.3). Sudden cardiac death free survival among patients experiencing VT was less than in those without (79% vs 92%; P = .02). After adjusting for gender, cardiac risk factors, and type of surgery, new-onset perioperative VT was associated with sudden cardiac death (hazard ratio [HR], 2.6; 95% confidence interval [CI], 1.1-5.8). CONCLUSION: Perioperative VT is likely to be associated with late sudden cardiac death and decreased survival. Continuous perioperative ECG is an effective method to identify VT and may allow improved management of these patients.

The efficacy and safety of clopidogrel in vascular surgery patients with immediate postoperative asymptomatic troponin T release for the prevention of late cardiac events: Rationale and design of the Dutch Echocardiographic Cardiac Risk Evaluation Applying Stress Echo-VII (DECREASE-VII) trial.

BACKGROUND: Major vascular surgery patients are at high risk for developing asymptomatic perioperative myocardial ischemia reflected by a postoperative troponin release without the presence of chest pain or electrocardiographic abnormalities. Long-term prognosis is severely compromised and characterized by an increased risk of long-term mortality and cardiovascular events. Current guidelines on perioperative care recommend single antiplatelet therapy with aspirin as prophylaxis for cardiovascular events. However, as perioperative surgical stress results in a prolonged hypercoagulable state, the postoperative addition of clopidogrel to aspirin within 7 days after perioperative asymptomatic cardiac ischemia could provide improved effective prevention for cardiovascular events. STUDY DESIGN: DECREASE-VII is a phase III, randomized, double-blind, placebo-controlled, multicenter clinical trial designed to evaluate the efficacy and safety of early postoperative dual antiplatelet therapy (aspirin and clopidogrel) for the prevention of cardiovascular events after major vascular surgery. Eligible patients undergoing a major vascular surgery (abdominal aorta or lower extremity vascular surgery) who developed perioperative asymptomatic troponin release are randomized 1:1 to clopidogrel or placebo (300-mg loading dose, followed by 75 mg daily) in addition to standard medical treatment with aspirin. The primary efficacy end point is the composite of cardiovascular death, stroke, or severe ischemia of the coronary or peripheral arterial circulation leading to an intervention. The evaluation of long-term safety includes bleeding defined by TIMI criteria. Recruitment began early 2010. The trial will continue until 750 patients are included and followed for at least 12 months. SUMMARY: DECREASE-VII is evaluating whether early postoperative dual antiplatelet therapy for patients developing asymptomatic cardiac ischemia after vascular surgery reduces cardiovascular events with a favorable safety profile.

Risk factors and outcome of new-onset cardiac arrhythmias in vascular surgery patients.

BACKGROUND: The pathophysiology of new-onset cardiac arrhythmias is complex and may bring about severe cardiovascular complications. The relevance of perioperative arrhythmias during vascular surgery has not been investigated. The aim of this study was to assess risk factors and prognosis of new-onset arrhythmias during vascular surgery. METHODS: A total of 513 vascular surgery patients, without a history of arrhythmias, were included. Cardiac risk factors, inflammatory status, and left ventricular function (LVF; N-terminal pro-B-type natriuretic peptide and echocardiography) were assessed. Continuous electrocardiography (ECG) recordings for 72 hours were used to identify ischemia and new-onset arrhythmias: atrial fibrillation, sustained ventricular tachycardia, supraventricular tachycardia, and ventricular fibrillation. Logistic regression analysis was applied to identify preoperative risk factors for arrhythmias. Cox regression analysis assessed the impact of arrhythmias on cardiovascular event-free survival during 1.7 years. RESULTS: New-onset arrhythmias occurred in 55 (11%) of 513 patients: atrial fibrillation, ventricular tachycardia, supraventricular tachycardia, and ventricular fibrillation occurred in 4%, 7%, 1%, and 0.2%, respectively. Continuous ECG showed myocardial ischemia and arrhythmias in 17 (3%) of 513 patients. Arrhythmia was preceded by ischemia in 10 of 55 cases. Increased age and reduced LVF were risk factors for the development of arrhythmias. Multivariate analysis showed that perioperative arrhythmias were associated with long-term cardiovascular events, irrespective of the presence of perioperative ischemia (hazard ratio 2.2, 95% CI 1.3-3.8, P = .004). CONCLUSION: New-onset perioperative arrhythmias are common after vascular surgery. The elderly and patients with reduced LVF show arrhythmias. Perioperative continuous ECG monitoring helps to identify this high-risk group at increased risk of cardiovascular events and death.

Prognostic implications of asymptomatic left ventricular dysfunction in patients undergoing vascular surgery.

BACKGROUND: The prognostic value of heart failure symptoms on postoperative outcome is well acknowledged in perioperative guidelines. The prognostic value of asymptomatic left ventricular (LV) dysfunction remains unknown. This study evaluated the prognostic implications of asymptomatic LV dysfunction in vascular surgery patients assessed with routine echocardiography. METHODS: Echocardiography was performed preoperatively in 1,005 consecutive vascular surgery patients. Systolic LV dysfunction was defined as LV ejection fraction less than 50%. Ratio of mitral-peak velocity during early and late filling, pulmonary vein flow, and deceleration time was used to diagnose diastolic LV dysfunction. Troponin-T measurements and electrocardiograms were performed routinely perioperatively. Multivariate regression analyses evaluated the relation between LV function and the study endpoints, 30-day cardiovascular events, and long-term cardiovascular mortality. RESULTS: Left ventricular dysfunction was diagnosed in 506 (50%) patients of which 80% were asymptomatic. In open vascular surgery (n = 649), both asymptomatic systolic and isolated diastolic LV dysfunctions were associated with 30-day cardiovascular events (odds ratios 2.3, 95% confidence interval [CI] 1.4-3.6 and 1.8, 95% CI 1.1-2.9, respectively) and long-term cardiovascular mortality (hazard ratios 4.6, 95% CI 2.4-8.5 and 3.0, 95% CI 1.5-6.0, respectively). In endovascular surgery (n = 356), only symptomatic heart failure was associated with 30-day cardiovascular events (odds ratio 1.8, 95% CI 1.1-2.9) and long-term cardiovascular mortality (hazard ratio 10.3, 95% CI 5.4-19.3). CONCLUSIONS: This study demonstrated that asymptomatic LV dysfunction is predictive for 30-day and long-term cardiovascular outcome in open vascular surgery patients. These data suggest that preoperative risk stratification should include not only solely heart failure symptoms but also routine preoperative echocardiography to risk stratify open vascular surgery patients.

Postoperative mortality in The Netherlands: a population-based analysis of surgery-specific risk in adults.

BACKGROUND: Few data are available that systematically describe rates and trends of postoperative mortality for fairly large, unselected patient populations. METHODS: This population-based study uses a registry of 3.7 million surgical procedures in 102 hospitals in The Netherlands during 1991-2005. Patients older than 20 yr who underwent an elective, nonday case, open surgical procedure were enrolled. Patient data included main (discharge) diagnosis, secondary diagnoses, dates of admission and discharge, death during admission, operations, age, sex, and a limited number of comorbidities classified according to the International Classification of Diseases 9th revision Clinical Modification. The main outcome measure was postoperative all-cause mortality. Univariable and multivariable logistic regression analyses were applied to evaluate the relationship between type of surgery and the main outcome. RESULTS: Postoperative all-cause death was observed in 67,879 patients (1.85%). In a model based on a classification into 11 main surgical categories, breast surgery was associated with lowest mortality (adjusted incidence, 0.07%), and vascular surgery was associated with highest mortality (adjusted incidence, 5.97%). In a model based on 36 surgical subcategories, the adjusted mortality ranged from 0.07% for hernia nuclei pulposus surgery to 18.5% for liver transplant. The c-index of the 36-category model was 0.88, which was significantly (P < 0.001) higher than the c-index that was associated with the simple surgical classification (low vs. high risk) in the commonly used Revised Cardiac Risk Index (c-index, 0.83). CONCLUSIONS: This population-based study provided a detailed and contemporary overview of postoperative mortality for the entire surgical spectrum, which may act as reference standard for surgical outcome in Western populations.

Impact of prophylactic beta-blocker therapy to prevent stroke after noncardiac surgery.

beta Blockers are widely used to improve the postoperative cardiac outcome in patients with coronary artery disease scheduled for noncardiac surgery. However, recently serious concerns regarding the safety of perioperative beta blockers have emerged. To assess the incidence, risk factors, and beta-blocker use associated with postoperative stroke in the Dutch Echocardiographic Cardiac Risk Evaluation Applying Stress Echocardiography (DECREASE) trials, we evaluated all 3,884 patients of the DECREASE trials for postoperative stroke. All cardiac risk factors and medication use were assessed. The incidence of stroke within 30 days after surgery was recorded. The incidence of postoperative stroke in the DECREASE trials was 0.46% (18 of 3,884). For the beta-blocker users, the incidence was 0.5%. All the strokes had an ischemic origin. A history of stroke was associated with a greater incidence of postoperative stroke (odds ratio [OR] 3.79, 95% confidence interval [CI] 1.2 to 11.6). Statins and anticoagulants were not associated with postoperative stroke (OR 0.85, 95% CI 0.3 to 2.4; and OR 1.27, 95% CI 0.4 to 4.6, respectively). No association with bisoprolol therapy was found (OR 1.16, 95% CI 0.4 to 3.4). In conclusion, with a low-dose bisoprolol regimen started > or =30 days before surgery, no association was observed between beta-blocker use and postoperative stroke.

Medication underuse during long-term follow-up in patients with peripheral arterial disease.

BACKGROUND: Patients with peripheral arterial disease constitute a high-risk population. Guideline-recommended medical therapy use is therefore of utmost importance. The aims of our study were to establish the patterns of guideline-recommended medication use in patients with PAD at the time of vascular surgery and after 3 years of follow up, and to evaluate the effect of these therapies on long-term mortality in this patient group. METHODS AND RESULTS: Data on 711 consecutive patients with peripheral arterial disease undergoing vascular surgery were collected from 11 hospitals in the Netherlands (enrollment between May and December 2004). After 3.1+/-0.1 years of follow-up, information on medication use was obtained by a questionnaire (n=465; 84% response rate among survivors). Guideline-recommended medical therapy use for the combination of aspirin and statins in all patients and beta-blockers in patients with ischemic heart disease was 41% in the perioperative period. The use of perioperative evidence-based medication was associated with a reduction of 3-year mortality after adjustment for clinical characteristics (hazard ratio, 0.65; 95% CI, 0.45 to 0.94). After 3 years of follow-up, aspirin was used in 74%, statins in 69%, and beta-blockers in 54% of the patients respectively. Guideline-recommended medical therapy use for the combination of aspirin, statins, and beta-blockers was 50%. CONCLUSIONS: The use of guideline recommended therapies in the perioperative period was associated with reduction in long-term mortality in patients with peripheral arterial disease. However, the proportion of patients receiving these evidence-based treatments-both at baseline and 3 years after vascular surgery-was lower than expected based on the current guidelines. These data highlight a clear opportunity to improve the quality of care in this high-risk group of patients.