Elise Kaspi

CD146 Short Isoform Increases the Proangiogenic Potential of Endothelial Progenitor Cells In Vitro and In Vivo

Rationale: CD146, a transmembrane immunoglobulin mainly expressed at the intercellular junction of endothelial cells, is involved in cell-cell cohesion, paracellular permeability, monocyte transmigration and angiogenesis. CD146 exists as 2 isoforms, short (sh) and long (lg), but which isoform is involved remains undefined. Objective: The recently described role of CD146 in angiogenesis prompted us to investigate which isoform was involved in this process in human late endothelial progenitors (EPCs), with the objective of increasing their proangiogenic potential. Methods and Results: Immunofluorescence experiments showed that, in subconfluent EPCs, shCD146 was localized in the nucleus and at the migrating edges of the membrane, whereas lgCD146 was intracellular. In confluent cells, shCD146 was redistributed at the apical membrane and lgCD146 was directed toward the junction. In contrast to lgCD146, shCD146 was overexpressed in EPCs as compared to mature endothelial cells and upregulated by vascular endothelial growth factor and SDF-1 (stromal cell–derived factor 1). Study of the properties of both isoforms in vitro provided evidence that shCD146 was involved in EPC adhesion to activated endothelium, migration, and proliferation, with a paracrine secretion of interleukin-8 or angiopoietin 2, whereas lgCD146 was implicated in stabilization of capillary-like structures in Matrigel and transendothelial permeability. In an animal model of hindlimb ischemia, transplantation of shCD146-modified EPCs selectively promoted both EPC engraftment and blood flow. Conclusions: Altogether, these findings establish that CD146 isoforms display distinct functions in vessels regeneration. Selective improvement of therapeutic angiogenesis by shCD146 overexpression suggests a potential interest of shCD146-transduced EPCs for the treatment of peripheral ischemic disease.

Soluble Melanoma Cell Adhesion Molecule (sMCAM/sCD146) Promotes Angiogenic Effects on Endothelial Progenitor Cells through Angiomotin

Background: Soluble melanoma cell adhesion molecule (sMCAM/sCD146) promotes angiogenic effects on endothelial progenitor cells (EPC). Results: sCD146 binds angiomotin in EPC and triggers the activation of different signaling pathways. Silencing angiomotin prevents this activation and angiogenic effects. Conclusion: Angiomotin is identified as a novel binding partner of sCD146. Significance: Angiomotin mediates the angiogenic effects of sCD146. The melanoma cell adhesion molecule (CD146) contains a circulating proteolytic variant (sCD146), which is involved in inflammation and angiogenesis. Its circulating level is modulated in different pathologies, but its intracellular transduction pathways are still largely unknown. Using peptide pulldown and mass spectrometry, we identified angiomotin as a sCD146-associated protein in endothelial progenitor cells (EPC). Interaction between angiomotin and sCD146 was confirmed by enzyme-linked immunosorbent assay (ELISA), homogeneous time-resolved fluorescence, and binding of sCD146 on both immobilized recombinant angiomotin and angiomotin-transfected cells. Silencing angiomotin in EPC inhibited sCD146 angiogenic effects, i.e. EPC migration, proliferation, and capacity to form capillary-like structures in Matrigel. In addition, sCD146 effects were inhibited by the angiomotin inhibitor angiostatin and competition with recombinant angiomotin. Finally, binding of sCD146 on angiomotin triggered the activation of several transduction pathways that were identified by antibody array. These results delineate a novel signaling pathway where sCD146 binds to angiomotin to stimulate a proangiogenic response. This result is important to find novel target cells of sCD146 and for the development of therapeutic strategies based on EPC in the treatment of ischemic diseases.

Detection of EpCAM-positive microparticles in pleural fluid: A new approach to mini-invasively identify patients with malignant pleural effusions

Pleural biomarkers allowing to mini-invasively discriminate benign from malignant pleural effusions are needed. Among potential candidates, microparticles (MPs) are extracellular vesicles that vectorize antigen derived from the parent cell. We hypothesized that tumor-derived MPs could be present in the pleural liquid and help to identify patients with malignant pleural effusions. Using highly sensitive flow cytometry and cryo-electron microscopy, we showed that large amounts of MPs from hematopoïetic and vascular origin could be detectable in pleural fluids. Their level did not differ between benign (n = 14) and malignant (n = 71) pleural effusions. Analysis of selected tumoral associated antigens (podoplanin, mucin 1 and EpCAM, epithelial-cell-adhesion-molecule) evidenced for the first time the presence of tumor-derived MPs expressing EpCAM in malignant pleural fluids only (Specificity = 93%, Sensitivity = 49% and 45% for flow cytometry and ELISA, respectively). The detection of EpCAM-positive-MPs (EpCAM + MPs) by flow cytometry showed a better specificity and sensitivity than ELISA to distinguish between pleural carcinoma and the others malignant pleural effusions (MPE; Sp: 96% vs 89%; Se: 79% vs 66%). Combining EpCAM+ MPs and cytology improved the diagnosis of MPE compared to cytology alone. This study establishes the basis for using EpCAM+ MPs as a promising new biomarker that could be added to the armamentarium to mini-invasively identify patients with malignant pleural effusions.

Identification of soluble CD146 as a regulator of trophoblast migration: potential role in placental vascular development

Both vasculogenesis and angiogenesis occur during normal placental vascular development. Additionally, the placenta undergoes a process of vascular mimicry (pseudo-vasculogenesis) where the placental extravillous trophoblast (EVT) that invade the spiral arteries convert from an epithelial to an endothelial phenotype during normal pregnancy. As soluble CD146 (sCD146) constitutes a new physiological factor with angiogenic properties, we hypothesized that it could be involved in the regulation of placental vascular development by acting on EVT. Using placental villous explants, we demonstrated that sCD146 inhibits EVT outgrowth. Consistently, we showed that sCD146 inhibits the ability of EVT cells (HTR8/SVneo) to migrate, invade and form tubes in Matrigel, without affecting their proliferation or apoptosis. The involvement of sCD146 in human pregnancy was investigated by evaluation of sCD146 levels in 50 pregnant women. We observed physiological down-regulation of sCD146 throughout pregnancy. These results prompted us to investigate the effect of prolonged sCD146 administration in a rat model of pregnancy. Repeated systemic sCD146 injections after coupling caused a significant decrease of pregnancy rate and number of embryos. Histological studies performed on placenta evidenced a reduced migration of glycogen cells (analogous to EVT in rat) in sCD146-treated rats. We propose that in human, sCD146 could represent both an attractive biomarker of placental vascular development and a therapeutic target in pregnancy complications associated with pathological angiogenesis.

HIV Protease Inhibitors Do Not Cause the Accumulation of Prelamin A in PBMCs from Patients Receiving First Line Therapy: The ANRS EP45 “Aging” Study

Background The ANRS EP45 “Aging” study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The present report focuses on lamin A processing, a pathway known to be altered in systemic genetic progeroid syndromes. Methods 35 HIV-1 infected patients being treated with first line antiretroviral therapy (ART, mean duration at inclusion: 2.7±1.3 years) containing boosted protease inhibitors (PI/r) (comprising lopinavir/ritonavir in 65% of patients) were recruited together with 49 seronegative age- and sex-matched control subjects (http://clinicaltrials.gov/, NCT01038999). In more than 88% of patients, the viral load was <40 copies/ml and the CD4+ cell count was >500/mm3. Prelamin A processing in peripheral blood mononuclear cells (PBMCs) from patients and controls was analysed by western blotting at inclusion. PBMCs from patients were also investigated at 12 and 24 months after enrolment in the study. PBMCs from healthy controls were also incubated with boosted lopinavir in culture medium containing various concentrations of proteins (4 to 80 g/L). Results Lamin A precursor was not observed in cohort patient PBMC regardless of the PI/r used, the dose and the plasma concentration. Prelamin A was detected in PBMC incubated in culture medium containing a low protein concentration (4 g/L) but not in plasma (60–80 g/L) or in medium supplemented with BSA (40 g/L), both of which contain a high protein concentration. Conclusions Prelamin A processing abnormalities were not observed in PBMCs from patients under the PI/r first line regimen. Therefore, PI/r do not appear to contribute to lamin A-related aging in PBMCs. In cultured PBMCs from healthy donors, prelamin A processing abnormalities were only observed when the protein concentration in the culture medium was low, thus increasing the amount of PI available to enter cells. ClinicalTrials.gov NCT01038999 http://clinicaltrials.gov/ct2/show/NCT01038999.

HIV-1 Infection and First Line ART Induced Differential Responses in Mitochondria from Blood Lymphocytes and Monocytes: The ANRS EP45 “Aging” Study

Background The ANRS EP45 “Aging” study investigates the cellular mechanisms involved in the accelerated aging of HIV-1 infected and treated patients. The data reported focus on mitochondria, organelles known to be involved in cell senescence. Methods 49 HIV-1 infected patients untreated with antiretroviral therapy, together with 49 seronegative age- and sex-matched control subjects and 81 HIV-1 infected and treated patients, were recruited by 3 AIDS centres (Marseille, Montpellier, Nice; France; http://clinicaltrials.gov/, NCT01038999). In more than 88% of treated patients, the viral load was <40 copies/ml and the CD4+ cell count was >500/mm3. ROS (reactive oxygen species) production and ΔΨm (inner membrane potential) were measured by flow cytometry in blood lymphocytes and monocytes (functional parameters). Three mitochondrial network quantitative morphological parameters were computed using confocal microscopy and image analysis. Three PBMC mitochondrial proteins (porin and subunits 2 and 4 of cytochrome C oxidase encoded by mtDNA or nuclear DNA, respectively) were analysed by western blotting. Results Quantitative changes in PBMC mitochondrial proteins were not induced by either HIV-1 infection or ART. Discriminant analysis integrating functional (ROS production and ΔΨm) or morphological (network volume density, fragmentation and branching) parameters revealed HIV-1 infection and ART differential effects according to cell type. First line ART tended to rescue lymphocyte mitochondrial parameters altered by viral infection, but induced slight changes in monocytes. No statistical difference was found between the effects of three ART regimens on mitochondrial parameters. Correlations between functional parameters and viral load confirmed the damaging effects of HIV-1 in lymphocyte mitochondria. Conclusions In patients considered to be clinically stable, mitochondria exhibited functional and morphological modifications in PBMCs resulting from either direct or indirect effects of HIV-1 infection (lymphocytes), or from first line ART (monocytes). Together with other tissue impairments, these changes may contribute to global aging. Trial Registration ClinicalTrials.gov NCT01038999 NCT01038999

Low lamin A expression in lung adenocarcinoma cells from pleural effusions is a pejorative factor associated with high number of metastatic sites and poor Performance status

The type V intermediate filament lamins are the principal components of the nuclear matrix, including the nuclear lamina. Lamins are divided into A-type and B-type, which are encoded by three genes, LMNA, LMNB1, and LMNB2. The alternative splicing of LMNA produces two major A-type lamins, lamin A and lamin C. Previous studies have suggested that lamins are involved in cancer development and progression. A-type lamins have been proposed as biomarkers for cancer diagnosis, prognosis, and/or follow-up. The aim of the present study was to investigate lamins in cancer cells from metastatic pleural effusions using immunofluorescence, western blotting, and flow cytometry. In a sub-group of lung adenocarcinomas, we found reduced expression of lamin A but not of lamin C. The reduction in lamin A expression was correlated with the loss of epithelial membrane antigen (EMA)/MUC-1, an epithelial marker that is involved in the epithelial to mesenchymal transition (EMT). Finally, the lamin A expression was inversely correlated with the number of metastatic sites and the WHO Performance status, and association of pleural, bone and lung metastatic localizations was more frequent when lamin A expression was reduced. In conclusion, low lamin A but not lamin C expression in pleural metastatic cells could represent a major actor in the development of metastasis, associated with EMT and could account for a pejorative factor correlated with a poor Performance status.

Positive pleural cytology is an indicator for visceral pleural invasion in metastatic pleural effusions

In case of undiagnosed pleural effusions, it is necessary to conduct thoracentesis with pleural fluid (PF) cytology. Yet, sensitivity of PF cytology is widely variable as a result of sample size, experience, and preparation method.

MicroRNAs in hereditary and sporadic premature aging syndromes and other laminopathies

Hereditary and sporadic laminopathies are caused by mutations in genes encoding lamins, their partners, or the metalloprotease ZMPSTE24/FACE1. Depending on the clinical phenotype, they are classified as tissue‐specific or systemic diseases. The latter mostly manifest with several accelerated aging features, as in Hutchinson–Gilford progeria syndrome (HGPS) and other progeroid syndromes. MicroRNAs are small noncoding RNAs described as powerful regulators of gene expression, mainly by degrading target mRNAs or by inhibiting their translation. In recent years, the role of these small RNAs has become an object of study in laminopathies using in vitro or in vivo murine models as well as cells/tissues of patients. To date, few miRNAs have been reported to exert protective effects in laminopathies, including miR‐9, which prevents progerin accumulation in HGPS neurons. The recent literature has described the potential implication of several other miRNAs in the pathophysiology of laminopathies, mostly by exerting deleterious effects. This review provides an overview of the current knowledge of the functional relevance and molecular insights of miRNAs in laminopathies. Furthermore, we discuss how these discoveries could help to better understand these diseases at the molecular level and could pave the way toward identifying new potential therapeutic targets and strategies based on miRNA modulation.

Chronic use of proton pump inhibitors, adverse events and potential biological mechanisms: A translational analysis

Proton pump inhibitors (PPIs) are among the most frequently prescribed drugs. Even if PPI are usually considered as safe, there is a growing concern for a range of adverse effects of chronic PPI therapy often in the absence of appropriate indications. We propose, after a summary of renal, cardiovascular and neurological complications (dementia, chronic kidney disease, myocardial infarction and stroke), an integrative overview of the potential biological mechanisms involved. Eleven positive pharmacoepidemiological studies, mainly based on health insurance database linkage to hospital database, reported an increased risk of complications associated to PPI use and often a graded association suggesting also a possible dose-response relationship. Several mechanisms have been suggested through in vitro studies (endothelial dysfunction, endothelial senescence, hypomagnesemia, increase of chromogranin A levels, decrease of nitric oxide in endothelial cells) leading to the impairment of vascular homesostasis, paving the way to these complications. Evidence that PPIs may have off-targets and pleiotropic effects are mounting and may impose a cautious attitude in the prescription of PPIs, especially in elderly and/or in the context of chronic use.