Enhancement of autophagic flux after neonatal cerebral hypoxia-ischemia and its region-specific relationship to apoptotic mechanisms.

The multiplicity of cell death mechanisms induced by neonatal hypoxia-ischemia makes neuroprotective treatment against neonatal asphyxia more difficult to achieve. Whereas the roles of apoptosis and necrosis in such conditions have been studied intensively, the implication of autophagic cell death has only recently been considered. Here, we used the most clinically relevant rodent model of perinatal asphyxia to investigate the involvement of autophagy in hypoxic-ischemic brain injury. Seven-day-old rats underwent permanent ligation of the right common carotid artery, followed by 2 hours of hypoxia. This condition not only increased autophagosomal abundance (increase in microtubule-associated protein 1 light chain 3-11 level and punctuate labeling) but also lysosomal activities (cathepsin D, acid phosphatase, and beta-N-acetylhexosaminidase) in cortical and hippocampal CA3-damaged neurons at 6 and 24 hours, demonstrating an increase in the autophagic flux. In the cortex, this enhanced autophagy may be related to apoptosis since some neurons presenting a high level of autophagy also expressed apoptotic features, including cleaved caspase-3. On the other hand, enhanced autophagy in CA3 was associated with a more purely autophagic cell death phenotype. In striking contrast to CA3 neurons, those in CA1 presented only a minimal increase in autophagy but strong apoptotic characteristics. These results suggest a role of enhanced autophagy in delayed neuronal death after severe hypoxia-ischemia that is differentially linked to apoptosis according to the cerebral region.

Lufaxin, a novel factor Xa inhibitor from the salivary gland of the sand fly Lutzomyia longipalpis blocks protease-activated receptor 2 activation and inhibits inflammation and thrombosis in vivo.

OBJECTIVE: Blood-sucking arthropods' salivary glands contain a remarkable diversity of antihemostatics. The aim of the present study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. METHODS AND RESULTS: Several L. longipalpis salivary proteins were expressed in human embryonic kidney 293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, noncompetitive, and reversible inhibitor of factor Xa (FXa). Notably, Lufaxin's primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, Dansyl-Glu-Gly-Arg-FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both prothrombin time and activated partial thromboplastin time. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with an equilibrium constant ≈3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents protease-activated receptor 2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl(3)-induced carotid artery thrombus formation and prolongs activated partial thromboplastin time ex vivo, implying that it works as an anticoagulant in vivo. Finally, salivary gland of sand flies was found to inhibit FXa and to interact with the enzyme. CONCLUSIONS: Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and anti-inflammatory activities. It is a useful tool to understand FXa structural features and its role in prohemostatic and proinflammatory events.

Noninvasive detection of macrophage-rich atherosclerotic plaque in hyperlipidemic rabbits using "positive contrast" magnetic resonance imaging.

OBJECTIVES: This study was designed to identify macrophage-rich atherosclerotic plaque noninvasively by imaging the tissue uptake of long-circulating superparamagnetic nanoparticles with a positive contrast off-resonance imaging sequence (inversion recovery with ON-resonant water suppression [IRON]). BACKGROUND: The sudden rupture of macrophage-rich atherosclerotic plaques can trigger the formation of an occlusive thrombus in coronary vessels, resulting in acute myocardial infarction. Therefore, a noninvasive technique that can identify macrophage-rich plaques and thereby assist with risk stratification of patients with atherosclerosis would be of great potential clinical utility. METHODS: Experiments were conducted on a clinical 3-T magnetic resonance imaging (MRI) scanner in 7 heritable hyperlipidemic and 4 control rabbits. Monocrystalline iron-oxide nanoparticles (MION)-47 were administrated intravenously (2 doses of 250 mumol Fe/kg), and animals underwent serial IRON-MRI before injection of the nanoparticles and serially after 1, 3, and 6 days. RESULTS: After administration of MION-47, a striking signal enhancement was found in areas of plaque only in hyperlipidemic rabbits. The magnitude of enhancement on magnetic resonance images had a high correlation with the number of macrophages determined by histology (p < 0.001) and allowed for the detection of macrophage-rich plaque with high accuracy (area under the curve: 0.92, SE: 0.04, 95% confidence interval: 0.84 to 0.96, p < 0.001). No significant signal enhancement was measured in remote areas without plaque by histology and in control rabbits without atherosclerosis. CONCLUSIONS: Using IRON-MRI in conjunction with superparamagnetic nanoparticles is a promising approach for the noninvasive evaluation of macrophage-rich, vulnerable plaques.

Predictive models of syncope causes in an outpatient clinic

The investigation of unexplained syncope remains a challenging clinical problem. In the present study we sought to evaluate the diagnostic value of a standardized work-up focusing on non invasive tests in patients with unexplained syncope referred to a syncope clinic, and whether certain combinations of clinical parameters are characteristic of rhythmic and reflex causes of syncope. METHODS AND RESULTS: 317 consecutive patients underwent a standardized work-up including a 12-lead ECG, physical examination, detailed history with screening for syncope-related symptoms using a structured questionnaire followed by carotid sinus massage (CSM), and head-up tilt test. Invasive testings including an electrophysiological study and implantation of a loop recorder were only performed in those with structural heart disease or traumatic syncope. Our work-up identified an etiology in 81% of the patients. Importantly, three quarters of the causes were established non invasively combining head-up tilt test, CSM and hyperventilation testing. Invasive tests yielded an additional 7% of diagnoses. Logistic analysis identified age and number of significant prodromes as the only predictive factors of rhythmic syncope. The same two factors, in addition to the duration of the ECG P-wave, were also predictive of vasovagal and psychogenic syncope. These factors, optimally combined in predictive models, showed a high negative and a modest positive predictive value. CONCLUSION: A standardized work-up focusing on non invasive tests allows to establish more than three quarters of syncope causes. Predictive models based on simple clinical parameters may help to distinguish between rhythmic and other causes of syncope

Extracorporeal membrane oxygenation support in acardia.

In extreme situations, such as hyperacute rejection of heart transplant or major heart trauma, heart preservation may not be possible. Our experimental team works on a project of peripheral extracorporeal membrane oxygenation (ECMO) support in acardia as a bridge to heart transplantation or artificial heart implantation. An ECMO support was established in five calves (58.6 ± 6.9 kg) by the transjugular insertion to the caval axis of a self-expanded cannula, with carotid artery return. After baseline measurements, ventricular fibrillation was induced, great arteries were clamped, heart was excised, and right and left atria remnants, containing pulmonary veins, were sutured together leaving an atrial septal defect over the caval axis cannula. Measurements of pump flow and arterial pressure were taken with the pulmonary artery clamped and anastomosed with the caval axis for a total of 6 hours. Pulmonary artery anastomosis to the caval axis provided an acceptable 6 hour hemodynamic stability, permitting a peripheral access ECMO support in extreme scenarios indicating a heart explantation.

Resistance of the internal mammary artery to restenosis: a histomorphologic study of various porcine arteries.

BACKGROUND/AIMS: Restenosis after percutaneous transluminal angioplasty (PTA) of the internal mammary artery (IMA) grafts is much less pronounced than in other arteries and venous grafts. The aim of the study was to test whether various arteries respond differently to dilatation. METHODS: PTA of the IMA, carotid, renal and circumflex coronary (RCx) arteries was performed in 9 pigs (balloon to artery ratio of 1:1.5). After 8 weeks, angiography was repeated and vessels prepared for histological analysis. Immunohistochemical staining was done to examine proliferative activity (Ki67) and to identify the vasa vasorum of the adventitia (F VIII-RA). RESULTS: The intima-media ratio after PTA was lowest in the IMA (0.06), followed by the carotid (0.27) and renal arteries (0.49) and the RCx (0.69). Proliferation of the intima was seen at 287 degrees of the vessel circumference in the RCx, at 286 degrees in the renal and at 166 degrees in the carotid artery. No proliferative activity was seen in the IMA. The intima-adventitia ratio was lower in the IMA than in the RCx and renal arteries (p &lt; 0.05). CONCLUSION: Intima proliferation after PTA varies between the different vessels, with best results seen in the IMA. There are differences in remodeling after PTA between muscular, muscular/elastic and elastic arteries.

Etiology of first-ever ischaemic stroke in European young adults: the 15 cities young stroke study.

BACKGROUND AND PURPOSE: Risk factors for IS in young adults differ between genders and evolve with age, but data on the age- and gender-specific differences by stroke etiology are scare. These features were compared based on individual patient data from 15 European stroke centers. METHODS: Stroke etiology was reported in detail for 3331 patients aged 15-49 years with first-ever IS according to Trial of Org in Acute Stroke Treatment (TOAST) criteria: large-artery atherosclerosis (LAA), cardioembolism (CE), small-vessel occlusion (SVO), other determined etiology, or undetermined etiology. CE was categorized into low- and high-risk sources. Other determined group was divided into dissection and other non-dissection causes. Comparisons were done using logistic regression, adjusting for age, gender, and center heterogeneity. RESULTS: Etiology remained undetermined in 39.6%. Other determined etiology was found in 21.6%, CE in 17.3%, SVO in 12.2%, and LAA in 9.3%. Other determined etiology was more common in females and younger patients, with cervical artery dissection being the single most common etiology (12.8%). CE was more common in younger patients. Within CE, the most frequent high-risk sources were atrial fibrillation/flutter (15.1%) and cardiomyopathy (11.5%). LAA, high-risk sources of CE, and SVO were more common in males. LAA and SVO showed an increasing frequency with age. No significant etiologic distribution differences were found amongst southern, central, or northern Europe. CONCLUSIONS: The etiology of IS in young adults has clear gender-specific patterns that change with age. A notable portion of these patients remains without an evident stroke mechanism according to TOAST criteria.

Blood glucose level on postoperative day 1 is predictive of adverse outcomes after cardiovascular surgery

AIM: Hyperglycaemia is now a recognized predictive factor of morbidity and mortality after coronary artery bypass grafting (CABG). For this reason, we aimed to evaluate the postoperative management of glucose control in patients undergoing cardiovascular surgery, and to assess the impact of glucose levels on in-hospital mortality and morbidity. METHODS: This was a retrospective study investigating the association between postoperative blood glucose and outcomes, including death, post-surgical complications, and length of stay in the intensive care unit (ICU) and in hospital. RESULTS: A total of 642 consecutive patients were enrolled into the study after cardiovascular surgery (CABG, carotid endarterectomy and bypass in the lower limbs). Patients' mean age was 68+/-10 years, and 74% were male. In-hospital mortality was 5% in diabetic patients vs 2% in non-diabetic patients (OR: 1.66, P=0.076). Having blood glucose levels in the upper quartile range (> or =8.8 mmol/L) on postoperative day 1 was independently associated with death (OR: 10.16, P=0.0002), infectious complications (OR: 1.76, P=0.04) and prolonged ICU stay (OR: 3.10, P<0.0001). Patients presenting with three or more hypoglycaemic episodes (<4.1 mmol/L) had increased rates of mortality (OR: 9.08, P<0.0001) and complications (OR: 8.57, P<0.0001). CONCLUSION: Glucose levels greater than 8.8 mmol/L on postoperative day 1 and having three or more hypoglycaemic episodes in the postoperative period were predictive of mortality and morbidity among patients undergoing cardiovascular surgery. This suggests that a multidisciplinary approach may be able to achieve better postoperative blood glucose control. Conclusion: Objectif: L'hyperglycémie a été reconnue comme facteur prédictif de morbidité et mortalité après un pontage aortocoronaire. Notre étude avait pour objectif d'évaluer la prise en charge postopératoire des glycémies chez les patients qui avaient subi une intervention chirurgicale cardiovasculaire et d'évaluer l'impact de ces glycémies sur la mortalité et la morbidité intrahospitalières. Méthodes: Étude rétrospective recherchant une association entre la glycémie postopératoire et les complications postchirurgicales, la mortalité et la durée du séjour aux soins intensifs et à l'hôpital. Résultats: L'étude a été réalisée sur 642 patients qui avaient subi une intervention chirurgicale cardiovasculaire (ex. pontage aortocoronaire, endartérectomie de la carotide, pontage artériel des membres inférieurs). L'âge moyen est de 68 ± 10 ans et 74 % des patients étaient de sexe masculin. La mortalité intrahospitalière a été de 5 % parmi les patients diabétiques et 2 % chez les non-diabétiques (OR 1,66, p = 0,076). Les taux de glycémies situés dans le quartile supérieur (≥ 8,8 mmol/l) à j1 postopératoire sont associés de manière indépendante avec la mortalité (OR 10,16, 95 % CI 3,20-39,00, p = 0,0002), les complications infectieuses (OR 1,76, 95 % CI 1,02-3,00, p = 0,04) et la durée du séjour aux soins intensifs (OR 3,10, 95 % CI 1,83-5,38, p < 0,0001). Les patients qui avaient présenté trois hypoglycémies ou plus (< 4,1 mmol/l) ont présenté un taux augmenté de mortalité (OR 9,08, p ≤ 0,0001) et de complications (OR 8,57, p < 0,0001). Conclusion : Des glycémies supérieures à 8,8 mmol/l à j1 postopératoire et la présence de trois hypoglycémies ou plus en période postopératoire sont des facteurs prédictifs de mauvais pronostic chez les patients qui avaient subi une intervention chirurgicale cardiovasculaire. Ainsi, une approche multidisciplinaire devrait être proposée afin d'obtenir un meilleur contrôle postopératoire des glycémies.

Late outcome of spinal cord stimulation for unreconstructable and limb-threatening lower limb ischemia.

OBJECTIVES: To determine whether the initial benefits of spinal cord stimulation (SCS) treatment for critical limb ischemia (CLI) persist over years. DESIGN: Analysis of data prospectively collected for every CLI patient receiving permanent SCS. Follow-up range 12 to 98 months (mean 46+/-23, median 50 months). POPULATION: 87 patients (28% stage III, 72%stage IV) with unreconstructable CLI due (83%) or not (17%) to atherosclerosis and with an initial sitting/supine transcutaneous pO2 gradient >15 mmHg. METHODS: Assessment of actuarial patient survival (PS), limb salvage (LS) and amputation-free patient survival (AFPS). Analysis of the impact of 15 risk factors on long-term outcomes using the Fischer's exact test for categorical variables and the t test for continuous variables. RESULTS: Follow-up was complete for patient and limb survival. A single non-atherosclerotic patient died during follow-up. Among atherosclerotic patients PS decreased from 88% at 1y, to 76% at 3y, 64% at 5y and 57% at 7y. LS reached 84% at 1y, 78% at 2y, 75% at 3y and remained stable thereafter. Diabetes was found to affect LS (p<0.05) and heart disease to reduce PS (p<0.01). AFPS was reduced in heart patients (p<0.01), diabetics (p<0.05) and in patients with previous stroke (p<0.05). CONCLUSIONS: In CLI patients the beneficial effects of SCS persist far beyond the first year of treatment and major amputation becomes infrequent after the second year.

Connexin 37 limits thrombus propensity by downregulating platelet reactivity.

Background- Formation of platelet plug initiates hemostasis after vascular injury and triggers thrombosis in ischemic disease. However, the mechanisms leading to the formation of a stable thrombus are poorly understood. Connexins comprise a family of proteins that form gap junctions enabling intercellular coordination of tissue activity, a process termed gap junctional intercellular communication. Methods and Results- In the present study, we show that megakaryocytes and platelets express connexin 37 (Cx37). Deletion of the Cx37 gene in mice shortens bleeding time and increases thrombus propensity. Aggregation is increased in murine Cx37(-/-) platelets or in murine Cx37(+/+) and human platelets treated with gap junction blockers. Intracellular microinjection of neurobiotin, a Cx37-permeant tracer, revealed gap junctional intercellular communication in platelet aggregates, which was impaired in Cx37(-/-) platelets and in human platelets exposed to gap junction blockers. Finally, healthy subjects homozygous for Cx37-1019C, a prognostic marker for atherosclerosis, display increased platelet responses compared with subjects carrying the Cx37-1019T allele. Expression of these polymorphic channels in communication-deficient cells revealed a decreased permeability of Cx37-1019C channels for neurobiotin. Conclusions- We propose that the establishment of gap junctional communication between Cx37-expressing platelets provides a mechanism to limit thrombus propensity. To our knowledge, these data provide the first evidence incriminating gap junctions in the pathogenesis of thrombosis.

Interleukin-8 secretion by fibroblasts induced by low density lipoproteins is p38 MAPK-dependent and leads to cell spreading and wound closure.

We have previously reported (Dobreva, I., Waeber, G., Mooser, V., James, R. W., and Widmann, C. (2003) J. Lipid Res. 44, 2382-2390) that low density lipoproteins (LDLs) induce activation of the p38 MAPK pathway, resulting in fibroblast spreading and lamellipodia formation. Here, we show that LDL-stimulated fibroblast spreading and wound sealing are due to secretion of a soluble factor. Using an antibody-based human protein array, interleukin-8 (IL-8) was identified as the main cytokine whose concentration was increased in supernatants from LDL-stimulated cells. Incubation of supernatants from LDL-treated cells with an anti-IL-8 blocking antibody completely abolished their ability to induce cell spreading and mediate wound closure. In addition, fibroblasts treated with recombinant IL-8 spread to the same extent as cells incubated with LDL or supernatants from LDL-treated cells. The ability of LDL and IL-8 to induce fibroblast spreading was mediated by the IL-8 receptor type II (CXCR-2). Furthermore, LDL-induced IL-8 production and subsequent wound closure required the activation of the p38 MAPK pathway, because both processes were abrogated by a specific p38 inhibitor. Therefore, the capacity of LDLs to induce fibroblast spreading and accelerate wound closure relies on their ability to stimulate IL-8 secretion in a p38 MAPK-dependent manner. Regulation of fibroblast shape and migration by lipoproteins may be relevant to atherosclerosis that is characterized by increased LDL cholesterol levels, IL-8 production, and extensive remodeling of the vessel wall.

Prediction of recanalization in acute stroke patients receiving intravenous and endovascular revascularization therapy.

BACKGROUND AND PURPOSE: The study aims to assess the recanalization rate in acute ischemic stroke patients who received no revascularization therapy, intravenous thrombolysis, and endovascular treatment, respectively, and to identify best clinical and imaging predictors of recanalization in each treatment group. METHODS: Clinical and imaging data were collected in 103 patients with acute ischemic stroke caused by anterior circulation arterial occlusion. We recorded demographics and vascular risk factors. We reviewed the noncontrast head computed tomographies to assess for hyperdense middle cerebral artery and its computed tomography density. We reviewed the computed tomography angiograms and the raw images to determine the site and degree of arterial occlusion, collateral score, clot burden score, and the density of the clot. Recanalization status was assessed on recanalization imaging using Thrombolysis in Myocardial Ischemia. Multivariate logistic regressions were utilized to determine the best predictors of outcome in each treatment group. RESULTS: Among the 103 study patients, 43 (42%) received intravenous thrombolysis, 34 (33%) received endovascular thrombolysis, and 26 (25%) did not receive any revascularization therapy. In the patients with intravenous thrombolysis or no revascularization therapy, recanalization of the vessel was more likely with intravenous thrombolysis (P = 0·046) and when M1/A1 was occluded (P = 0·001). In this subgroup of patients, clot burden score, cervical degree of stenosis (North American Symptomatic Carotid Endarterectomy Trial), and hyperlipidemia status added information to the aforementioned likelihood of recanalization at the patient level (P < 0·001). In patients with endovascular thrombolysis, recanalization of the vessel was more likely in the case of a higher computed tomography angiogram clot density (P = 0·012), and in this subgroup of patients gender added information to the likelihood of recanalization at the patient level (P = 0·044). CONCLUSION: The overall likelihood of recanalization was the highest in the endovascular group, and higher for intravenous thrombolysis compared with no revascularization therapy. However, our statistical models of recanalization for each individual patient indicate significant variability between treatment options, suggesting the need to include this prediction in the personalized treatment selection.

Arterial properties in relation to genetic variations in the adducin subunits in a white population.

BACKGROUND: Adducin is a membrane skeleton protein, which consists of either alpha- and beta- or alpha- and gamma-subunits. We investigated whether arterial characteristics might be related to the genes encoding ADD1 (Gly460Trp-rs4961), ADD2 (C1797T-rs4984), and ADD3 (IVS11+386A>G-rs3731566). METHODS: We randomly recruited 1,126 Flemish subjects (mean age, 43.8 years; 50.3% women). Using a wall-tracking ultrasound system, we measured the properties of the carotid, femoral, and brachial arteries. We studied multivariate-adjusted phenotype-genotype associations, using a population- and family-based approach. RESULTS: In single-gene analyses, brachial diameter was 0.15 mm (P = 0.0022) larger, and brachial distensibility and cross-sectional compliance were 1.55 x 10(-3)/kPa (P = 0.013) and 0.017 mm(2)/kPa (P = 0.0029) lower in ADD3 AA than ADD3 GG homozygotes with an additive effect of the G allele. In multiple-gene analyses, the association of brachial diameter and distensibility with the ADD3 G allele occurred only in ADD1 GlyGly homozygotes. Otherwise, the associations between the arterial phenotypes in the three vascular beds and the ADD1 or ADD2 polymorphisms were not significant. In family-based analyses, the multivariate-adjusted heritability was 0.52, 0.38, and 0.30 for brachial diameter, distensibility, and cross-sectional compliance, respectively (P < 0.001). There was no evidence for population stratification (0.07 < or = P < or = 0.96). Transmission of the mutated ADD3 G allele was associated with smaller brachial diameter in 342 informative offspring (-0.12 +/- 0.04 mm; P = 0.0085) and in 209 offspring, who were ADD1 GlyGly homozygotes (-0.14 +/- 0.06 mm; P = 0.018). CONCLUSIONS: In ADD1 GlyGly homozygotes, the properties of the brachial artery are related to the ADD3 (A386G) polymorphism, but the underlying mechanism needs further clarification.

Blood glucose level on postoperative day 1 is predictive of adverse outcomes after cardiovascular surgery.

AIM: Hyperglycaemia is now a recognized predictive factor of morbidity and mortality after coronary artery bypass grafting (CABG). For this reason, we aimed to evaluate the postoperative management of glucose control in patients undergoing cardiovascular surgery, and to assess the impact of glucose levels on in-hospital mortality and morbidity. METHODS: This was a retrospective study investigating the association between postoperative blood glucose and outcomes, including death, post-surgical complications, and length of stay in the intensive care unit (ICU) and in hospital. RESULTS: A total of 642 consecutive patients were enrolled into the study after cardiovascular surgery (CABG, carotid endarterectomy and bypass in the lower limbs). Patients' mean age was 68+/-10 years, and 74% were male. In-hospital mortality was 5% in diabetic patients vs 2% in non-diabetic patients (OR: 1.66, P=0.076). Having blood glucose levels in the upper quartile range (> or =8.8 mmol/L) on postoperative day 1 was independently associated with death (OR: 10.16, P=0.0002), infectious complications (OR: 1.76, P=0.04) and prolonged ICU stay (OR: 3.10, P<0.0001). Patients presenting with three or more hypoglycaemic episodes (<4.1 mmol/L) had increased rates of mortality (OR: 9.08, P<0.0001) and complications (OR: 8.57, P<0.0001). CONCLUSION: Glucose levels greater than 8.8 mmol/L on postoperative day 1 and having three or more hypoglycaemic episodes in the postoperative period were predictive of mortality and morbidity among patients undergoing cardiovascular surgery. This suggests that a multidisciplinary approach may be able to achieve better postoperative blood glucose control.