Loïc Doeuvre

Hippocampal subfield volumetry in mild cognitive impairment, Alzheimer's disease and semantic dementia.

Background Hippocampal atrophy is a well-known feature of Alzheimers disease (AD), but sensitivity and specificity of hippocampal volumetry are limited. Neuropathological studies have shown that hippocampal subfields are differentially vulnerable to AD; hippocampal subfield volumetry may thus prove to be more accurate than global hippocampal volumetry to detect AD. Methods CA1, subiculum and other subfields were manually delineated from 40 healthy controls, 18 AD, 17 amnestic Mild Cognitive Impairment (aMCI), and 8 semantic dementia (SD) patients using a previously developed high resolution MRI procedure. Non-parametric group comparisons and receiver operating characteristic (ROC) analyses were conducted. Complementary analyses were conducted to evaluate differences of hemispheric asymmetry and anterior-predominance between AD and SD patients and to distinguish aMCI patients with or without β-amyloid deposition as assessed by Florbetapir-TEP. Results Global hippocampi were atrophied in all three patient groups and volume decreases were maximal in the CA1 subfield (22% loss in aMCI, 27% in both AD and SD; all p < 0.001). In aMCI, CA1 volumetry was more accurate than global hippocampal measurement to distinguish patients from controls (areas under the ROC curve = 0.88 and 0.76, respectively; p = 0.05) and preliminary analyses suggest that it was independent from the presence of β-amyloid deposition. In patients with SD, whereas the degree of CA1 and subiculum atrophy was similar to that found in AD patients, hemispheric and anterior–posterior asymmetry were significantly more marked than in AD with greater involvement of the left and anterior hippocampal subfields. Conclusions The findings suggest that CA1 measurement is more sensitive than global hippocampal volumetry to detect structural changes at the pre-dementia stage, although the predominance of CA1 atrophy does not appear to be specific to AD pathophysiological processes.

Leukocyte- and endothelial-derived microparticles: a circulating source for fibrinolysis

Background We recently assigned a new fibrinolytic function to cell-derived microparticles in vitro. In this study we explored the relevance of this novel property of microparticles to the in vivo situation. Design and Methods Circulating microparticles were isolated from the plasma of patients with thrombotic thrombocytopenic purpura or cardiovascular disease and from healthy subjects. Microparticles were also obtained from purified human blood cell subpopulations. The plasminogen activators on microparticles were identified by flow cytometry and enzyme-linked immunosorbent assays; their capacity to generate plasmin was quantified with a chromogenic assay and their fibrinolytic activity was determined by zymography. Results Circulating microparticles isolated from patients generate a range of plasmin activity at their surface. This property was related to a variable content of urokinase-type plasminogen activator and/or tissue plasminogen activator. Using distinct microparticle subpopulations, we demonstrated that plasmin is generated on endothelial and leukocyte microparticles, but not on microparticles of platelet or erythrocyte origin. Leukocyte-derived microparticles bear urokinase-type plasminogen activator and its receptor whereas endothelial microparticles carry tissue plasminogen activator and tissue plasminogen activator/inhibitor complexes. Conclusions Endothelial and leukocyte microparticles, bearing respectively tissue plasminogen activator or urokinase-type plasminogen activator, support a part of the fibrinolytic activity in the circulation which is modulated in pathological settings. Awareness of this blood-borne fibrinolytic activity conveyed by microparticles provides a more comprehensive view of the role of microparticles in the hemostatic equilibrium.

Fibrinolytic cross-talk: a new mechanism for plasmin formation

Fibrinolysis and pericellular proteolysis depend on molecular coassembly of plasminogen and its activator on cell, fibrin, or matrix surfaces. We report here the existence of a fibrinolytic cross-talk mechanism bypassing the requirement for their molecular coassembly on the same surface. First, we demonstrate that, despite impaired binding of Glu-plasminogen to the cell membrane by epsilon-aminocaproic acid (epsilon-ACA) or by a lysine-binding site-specific mAb, plasmin is unexpectedly formed by cell-associated urokinase (uPA). Second, we show that Glu-plasminogen bound to carboxy-terminal lysine residues in platelets, fibrin, or extracellular matrix components (fibronectin, laminin) is transformed into plasmin by uPA expressed on monocytes or endothelial cell-derived microparticles but not by tissue-type plasminogen activator (tPA) expressed on neurons. A 2-fold increase in plasmin formation was observed over activation on the same surface. Altogether, these data indicate that cellular uPA but not tPA expressed by distinct cells is specifically involved in the recognition of conformational changes and activation of Glu-plasminogen bound to other biologic surfaces via a lysine-dependent mechanism. This uPA-driven cross-talk mechanism generates plasmin in situ with a high efficiency, thus highlighting its potential physiologic relevance in fibrinolysis and matrix proteolysis induced by inflammatory cells or cell-derived microparticles.

Cognitive and Brain Profiles Associated with Current Neuroimaging Biomarkers of Preclinical Alzheimer's Disease

Neuroimaging biomarkers, namely hippocampal volume loss, temporoparietal hypometabolism, and neocortical β-amyloid (Aβ) deposition, are included in the recent research criteria for preclinical Alzheimers disease (AD). However, how to use these biomarkers is still being debated, especially regarding their sequence. Our aim was to characterize the cognitive and brain profiles of elders classified as positive or negative for each biomarker to further our understanding of their use in the preclinical diagnosis of AD. Fifty-four cognitively normal individuals (age = 65.8 ± 8.3 years) underwent neuropsychological tests (structural MRI, FDG-PET, and Florbetapir-PET) and were dichotomized into positive or negative independently for each neuroimaging biomarker. Demographic, neuropsychological, and neuroimaging data were compared between positive and negative subgroups. The MRI-positive subgroup had lower executive performances and mixed patterns of lower volume and metabolism in AD-characteristic regions and in the prefrontal cortex. The FDG-positive subgroup showed only hypometabolism, predominantly in AD-sensitive areas extending to the whole neocortex, compared with the FDG-negative subgroup. The amyloid-positive subgroup was older and included more APOE ε4 carriers compared with the amyloid-negative subgroup. When considering MRI and/or FDG biomarkers together (i.e., the neurodegeneration-positive), there was a trend for an inverse relationship with Aβ deposition such that those with neurodegeneration tended to show less Aβ deposition and the reverse was true as well. Our findings suggest that: (1) MRI and FDG biomarkers provide complementary rather than redundant information and (2) relatively young cognitively normal elders tend to have either neurodegeneration or Aβ deposition, but not both, suggesting additive rather than sequential/causative links between AD neuroimaging biomarkers at this age. SIGNIFICANCE STATEMENT Neuroimaging biomarkers are included in the recent research criteria for preclinical Alzheimers disease (AD). However, how to use these biomarkers is still being debated, especially regarding their sequence. Our findings suggest that MRI and FDG-PET biomarkers should be used in combination, offering an additive contribution instead of reflecting the same process of neurodegeneration. Moreover, the present study also challenges the hierarchical use of the neuroimaging biomarkers in preclinical AD because it suggests that the neurodegeneration observed in this population is not due to β-amyloid deposition. Rather, our results suggest that β-amyloid- and tau-related pathological processes may interact but not necessarily appear in a systematic sequence.

Synergistic Effect of Thrombin and CD40 Ligand on Endothelial Matrix Metalloproteinase-10 Expression and Microparticle Generation In Vitro and In Vivo

Objective—Thrombin induces CD40 ligand (CD40L) and matrix metalloproteinases (MMPs) under inflammatory/prothrombotic conditions. Thrombin and CD40L could modulate endothelial MMP-10 expression in vitro and in vivo. Methods and Results—Human endothelial cells were stimulated with thrombin (0.1–10 U/mL), CD40L (0.25–1 &mgr;g/mL), or their combination (thrombin/CD40L) to assess MMP-10 expression and microparticle generation. Thrombin/CD40L elicited higher MMP-10 mRNA (5-fold; P<0.001) and protein levels (4.5-fold; P<0.001) than either stimulus alone. This effect was mimicked by a protease-activated receptor-1 agonist and antagonized by hirudin, a-protease-activated receptor-1, &agr;-CD40L, and &agr;-CD40 antibodies. The synergistic effect was dependent on p38 mitogen-activated protein kinase and c-Jun N-terminal kinase-1 pathways. Thrombin also upregulated the expression of CD40 in endothelial cell surface increasing its availability, thereby favoring its synergistic effects with CD40L. In mice, thrombin/CD40L further increased the aortic MMP-10 expression. Septic patients with systemic inflammation and enhanced thrombin generation (n=60) exhibited increased MMP-10 and soluble CD40L levels associated with adverse clinical outcome. Endothelial and systemic activation by thrombin/CD40L and lipopolysaccharide also increased microparticles harboring MMP-10 and CD40L. Conclusion—Thrombin/CD40L elicited a strong synergistic effect on endothelial MMP-10 expression and microparticles containing MMP-10 in vitro and in vivo, which may represent a new link between inflammation/thrombosis with prognostic implications.

Plasmin on adherent cells: from microvesiculation to apoptosis.

Cell activation by stressors is characterized by a sequence of detectable phenotypic cell changes. A given stimulus, depending on its strength, induces modifications in the activity of membrane phospholipid transporters and calpains, which lead to phosphatidylserine exposure, membrane blebbing and the release of microparticles (nanoscale membrane vesicles). This vesiculation could be considered as a warning signal that may be followed, if the stimulus is maintained, by cell detachment-induced apoptosis. In the present study, plasminogen incubated with adherent cells is converted into plasmin by constitutively expressed tPA (tissue-type plasminogen activator) or uPA (urokinase-type plasminogen activator). Plasmin formed on the cell membrane then induces a unique response characterized by membrane blebbing and vesiculation. Hitherto unknown for plasmin, these membrane changes are similar to those induced by thrombin on platelets. If plasmin formation persists, matrix proteins are then degraded, cells lose their attachments and enter the apoptotic process, characterized by DNA fragmentation and specific ultrastructural features. Since other proteolytic or inflammatory stimuli may evoke similar responses in different types of adherent cells, the proposed experimental procedure can be used to distinguish activated adherent cells from cells entering the apoptotic process. Such a distinction is crucial for evaluating the effects of mediators, inhibitors and potential therapeutic agents.

Plasminogen in cerebrospinal fluid originates from circulating blood

BackgroundPlasminogen activation is a ubiquitous source of fibrinolytic and proteolytic activity. Besides its role in prevention of thrombosis, plasminogen is involved in inflammatory reactions in the central nervous system. Plasminogen has been detected in the cerebrospinal fluid (CSF) of patients with inflammatory diseases; however, its origin remains controversial, as the blood-CSF barrier may restrict its diffusion from blood.MethodsWe investigated the origin of plasminogen in CSF using Alexa Fluor 488-labelled rat plasminogen injected into rats with systemic inflammation and blood-CSF barrier dysfunction provoked by lipopolysaccharide (LPS). Near-infrared fluorescence imaging and immunohistochemistry fluorescence microscopy were used to identify plasminogen in brain structures, its concentration and functionality were determined by Western blotting and a chromogenic substrate assay, respectively. In parallel, plasminogen was investigated in CSF from patients with Guillain-Barré syndrome (n = 15), multiple sclerosis (n = 19) and noninflammatory neurological diseases (n = 8).ResultsEndogenous rat plasminogen was detected in higher amounts in the CSF and urine of LPS-treated animals as compared to controls. In LPS-primed rats, circulating Alexa Fluor 488-labelled rat plasminogen was abundantly localized in the choroid plexus, CSF and urine. Plasminogen in human CSF was higher in Guillain-Barré syndrome (median = 1.28 ng/μl (interquartile range (IQR) = 0.66 to 1.59)) as compared to multiple sclerosis (median = 0.3 ng/μl (IQR = 0.16 to 0.61)) and to noninflammatory neurological diseases (median = 0.27 ng/μl (IQR = 0.18 to 0.35)).ConclusionsOur findings demonstrate that plasminogen is transported from circulating blood into the CSF of rats via the choroid plexus during inflammation. Our data suggest that a similar mechanism may explain the high CSF concentrations of plasminogen detected in patients with inflammation-derived CSF barrier impairment.