Objective-Cellular microparticles (MP) are promising biomarkers in many pathological situations. Although flow cytometry (FCM) is widely used for their measurement, it has raised controversies because the smallest MP size falls below the detection limit of standard FCM (sd-FCM). Following recent technological improvements leading to high sensitivity FCM (hs-FCM), our objectives were (1) to evaluate the potential of hs-FCM for extended MP detection, (2) to set up a standardized protocol for MP enumeration, and (3) to compare MP counts obtained with both sensitivity levels. Methods and Results-Compared with sd-FCM, hs-FCM displayed improved forward scatter resolution and lower background noise, allowing us to discriminate previously undetectable small MP in plasma samples. Using fluorescent beads with appropriate sizes (0.1/0.3/0.5/0.9 m) and relative amounts, a new standardized hs-FCM MP protocol was set up and provided reproducible MP counts. Applied to coronary patient samples, it resulted into 8-to 20-fold increases in MP counts as compared with sd-FCM. Interestingly, the ratio between small and large MP varied according to clinical status but also depending on MP subset, suggesting access to new biological information. 3-5 Circulating MP are elevated in many prothrombotic and inflammatory disorders; cardiovascular, autoimmune, and infectious diseases; and cancer. 4,[6][7][8][9][10] Although MP counts may provide useful diagnostic/prognostic information, assessment of their pathophysiological relevance is hampered by methodological concerns. Flow cytometry (FCM) is the most commonly used technique, 11 allowing both enumeration and characterization of MP cellular origin with high throughput. The forward scatter (FS) parameter is generally used to define the analysis gate for MP. Unfortunately, because of their limited FS sensitivity, standard flow cytometers measure only a small part of the MP population. Recently, high-sensitivity flow cytometers with significantly improved light scatter detection became available. As we first reported, these instruments resolve a previously undetectable subpopulation of small-size MPs. Conclusion-Recent12 Among the technical improvements found in high sensitivity FCM (hs-FCM), a new option of the Beckman Coulter Gallios called W2 increases FS resolution by amplifying the FS signals collected at large angles. 13 Whether this option can be used in a standardized manner, to improve the detection of MPs derived from platelets (PMP), erythrocytes (Ery-MP), leukocytes (Leu-MP), and endothelial cells (EMP), remains an open question. Our objectives were then (1) to evaluate the impact of high-sensitivity flow cytometer technical improvement on the detection of small-size MP, (2) to set up a new standardized protocol based on calibrated microbeads for MP enumeration, and (3) to compare MP counts obtained at both sensitivity levels of FCM. MethodsBlood and plasma samples, MP preparation and flow cytometric analysis, size-conditioned MP filtration, intra-and inter-instrument reproducibi...

Objectives-During inflammation, cell adhesion molecules are modulated or redistributed for leukocyte transmigration.Among molecules at the interendothelial junction, CD146 is involved in cell-cell cohesion and permeability, but its role in monocyte transmigration is unknown. Methods and Results-TNF enhanced CD146 expression at the junction and apical membrane of human umbilical veins endothelial cells (HUVECs) through CD146 synthesis and intracellular store redistribution. In addition, TNF increased the release of a soluble form (sCD146) through a metalloproteinase-dependent mechanism. The redistribution of CD146 to the junction led us to investigate its role in monocyte transmigration using THP1 and freshly isolated monocytes.Evidence that CD146 contributes to monocyte transmigration was provided by inhibition experiments using anti-CD146 antibodies and CD146 siRNA in HUVECs. In addition, sCD146 specifically bound both monocytes and HUVECs and dose-dependently increased monocyte transmigration. Assessment of sCD146 binding on immobilized CD146 failed to evidence any homophilic interaction. Together, our data suggest endothelial CD146 binds heterophilically with a yet unknown ligand on monocytes. Conclusions-Our results demonstrate that CD146 is regulated by the inflammatory cytokine TNF and that CD146 and sCD146 are both involved in monocyte transendothelial migration during inflammation. Key Words: endothelial cells Ⅲ inflammation Ⅲ cytokines Ⅲ human Ⅲ adhesion molecules T he endothelial junctions play a fundamental role in endothelial integrity, vascular permeability, and cellular traffic. 1 At least 2 types of cell-to-cell junctional structures have been identified in the endothelium: adherens junctions (AJ) and tight junctions (TJ). 2 These junctions are tightly regulated structures composed of several adhesion molecules interacting with cytoskeletal proteins. 3 Among the adhesive molecules, endothelial VE-cadherin 4 is localized in AJ and junctional adhesion molecule (JAM) 5 in TJ, whereas other molecules such as PECAM-1/CD31 (platelet endothelial cell adhesion molecule-1) and CD99 are not restricted to 1 type of junctional structure. 6 The inflammatory response is characterized by leukocyte infiltration from the circulation toward the tissues, after a multistep process in which proinflammatory endothelial activation results in increased vascular permeability and then in leukocyte adhesion and transmigration. 7 Endothelial activation is mediated by several inflammatory cytokines. Among them, TNF␣ increases the expression of cell adhesion molecules like ICAM-1 or VCAM-1 and induces the redistribution of junctional adhesion molecules such as PECAM-1, JAM, VE-cadherin, and CD99 which, in turn, promote the transendothelial migration of leukocytes. 8 We have previously shown that CD146 (S-Endo1 Ag) is a component of the endothelial junction localized outside defined junctional structures. 9 CD146, also referred to as MUC18, is a member of the immunoglobulin superfamily (IgSF) constitutively expressed in all typ...

Hutchinson–Gilford progeria syndrome (HGPS) is a lethal premature and accelerated aging disease caused by a de novo point mutation in LMNA encoding A‐type lamins. Progerin, a truncated and toxic prelamin A issued from aberrant splicing, accumulates in HGPS cells' nuclei and is a hallmark of the disease. Small amounts of progerin are also produced during normal aging. We show that progerin is sequestered into abnormally shaped promyelocytic nuclear bodies, identified as novel biomarkers in late passage HGPS cell lines. We found that the proteasome inhibitor MG132 induces progerin degradation through macroautophagy and strongly reduces progerin production through downregulation of SRSF‐1 and SRSF‐5 accumulation, controlling prelamin A mRNA aberrant splicing. MG132 treatment improves cellular HGPS phenotypes. MG132 injection in skeletal muscle of Lmna G609G/G609G mice locally reduces SRSF‐1 expression and progerin levels. Altogether, we demonstrate progerin reduction based on MG132 dual action and shed light on a promising class of molecules toward a potential therapy for children with HGPS.