Validation of reference genes for real-time PCR of cord blood mononuclear cells, differentiating endothelial progenitor cells, and mature endothelial cells

In the last ten years, endothelial progenitor cells (EPCs) have gained interest as an attractive cell population in regenerative medicine for vascular applications. This population is defined as the precursor of endothelial mature cells (ECs) through a process of differentiation. To our knowledge, no single marker can be used to discriminate them from mature ECs. To effectively study their differentiation kinetics, gene expression must be assessed. Quantitative real-time PCR (RT-qPCR) is widely used to analyze gene expression. To minimize the impact of variances from RT-qPCR, a rigorous selection of reference genes must be performed prior to any experiments due to variations in experimental conditions. In this study, CD34+ mononuclear cells were extracted from human cord blood and differentiated into EPCs after seeding for a maximum period of 21 days. To choose the best combinations of reference genes, we compared the results of EPCs, CD34+ mononuclear cells, and mature endothelial cells to ensure that the differentiation kinetics did not affect the expression of our selected reference genes. The expression levels of seven genes, namely, YWHAZ, GAPDH, HPRT1, RPLP0, UBC, B2M, and TBP were thus compared. The algorithms geNorm, NormFinder, BestKeeper, and the Comparative ΔCt method were employed to assess the expression of each candidate gene. Overall results reveal that the expression stability of reference genes may differ depending on the statistical program used. YWHAZ, GAPDH, and UBC composed the optimal set of reference genes for the gene expression studies performed by RT-qPCR in our experimental conditions. This work can thus serve as a starting point for the selection of candidate reference genes to normalize the levels of gene expression in endothelial progenitor cell population

Sizing nanomatter in biological fluids by fluorescence single particle tracking

Accurate sizing of nanoparticles in biological media is important for drug delivery and biomedical imaging applications since size directly influences the nanoparticle processing and nanotoxicity in vivo. Using fluorescence single particle cracking we have succeeded for the first time in following the aggregation of drug delivery nanoparticles in real time in undiluted whole blood. We demonstrate that, by using a suitable surface functionalization, nanoparticle aggregation in the blood circulation is prevented to a large extent

Fluorescence single particle tracking for sizing of nanoparticles in undiluted biological fluids

While extremely relevant to many life science fields, such as biomedical diagnostics and drug delivery, studies on the size of nanoparticulate matter dispersed in biofluids are missing due to a lack of suitable methods. Here we report that fluorescence single particle tracking (fSPT) with maximum entropy analysis is the first technique suited for accurate sizing of nanoparticles dispersed in biofluids, such as whole blood. After a thorough validation, the fSPT sizing method was applied to liposomes that have been under investigation for decades as nanocarriers for drugs. The tendency of these liposomes to form aggregates in whole blood was tested in vitro and in vivo. In addition, we have demonstrated that the fSPT sizing technique can be used for identifying and sizing natural cell-derived microparticles directly in plasma. fSPT sizing opens up the possibility to systematically study the size and aggregation of endogenous or exogenous nanoparticles in biofluids

Distinguishing Plasmin-Generating Microvesicles: Tiny Messengers Involved in Fibrinolysis and Proteolysis

A number of stressors and inflammatory mediators (cytokines, proteases, oxidative stress mediators) released during inflammation or ischemia stimulate and activate cells in blood, the vessel wall or tissues. The most well-known functional and phenotypic responses of activated cells are (1) the immediate expression and/or release of stored or newly synthesized bioactive molecules, and (2) membrane blebbing followed by release of microvesicles. An ultimate response, namely the formation of extracellular traps by neutrophils (NETs), is outside the scope of this work. The main objective of this article is to provide an overview on the mechanism of plasminogen reception and activation at the surface of cell-derived microvesicles, new actors in fibrinolysis and proteolysis. The role of microvesicle-bound plasmin in pathological settings involving inflammation, atherosclerosis, angiogenesis, and tumour growth, remains to be investigated. Further studies are necessary to determine if profibrinolytic microvesicles are involved in a finely regulated equilibrium with pro-coagulant microvesicles, which ensures a balanced haemostasis, leading to the maintenance of vascular patency

Fibrinolysis, new concepts and new mechanisms: fibrinolytic microvesicles and fibrinolytic crosstalk: Fibrinolysis, new concepts and new mechanisms

International audienceThrombus lysis is the consequence of a restricted number of reactions localised to the surface of fibrin. A functional defect or an insufficient fibrinolytic response may lead to thrombosis with severe or fatal clinical consequences, e.g. myocardial infarction and ischemic stroke. Despite this clinical exigency and a real progress in the knowledge of the different components of this system (plasminogen and its activators, inhibitors and receptors), its functional evaluation still remains a challenge in haemostasis. The absolute requirement of a template for molecular assembly of plasminogen and its activators (tissue- and urokinase-type plasminogen activators: tPA and uPA) restricts the formation of plasmin and protects its activity onto the surface of, respectively, fibrin and cells. In contrast, plasmin and tPA released from the clot during its lysis are immediately neutralised by their respective inhibitors α2-antiplasmin and plasminogen activator inhibitor 1, PAI-1). It seems therefore almost impossible to detect fibrinolytic activity in plasma with methods currently in use. Because of its unavailability, it is also impossible to measure the degree of fibrinolysis directly on the clot. Notwithstanding, it was recently discovered that circulating membrane microvesicles might be indicators of the fibrinolytic response to an inflammatory or prothrombotic process. These cell-derived fibrinolytic microvesicles bear at their membrane the plasminogen activators expressed by the parent cell: tPA from endothelial cells and uPA from leukocytes. These molecules are localised at the membrane surface and have the capacity to activate plasminogen into plasmin in situ. Moreover, it was recently discovered that these microvesicles might participate in a new mechanism of plasmin formation requiring a cross-talk between two different surfaces. In this fibrinolytic cross-talk one of the surfaces bear plasminogen (fibrin, extracellular matrix or platelets) whereas the other surface carry the plasminogen activator, typically leukocyte-derived microvesicles bearing uPA. These new actors and concepts in plasminogen activation represent hitherto unknown pathways in our comprehension of fibrinolysis and potential novel biomarkers in clinical practice

Membrane microvesicles: a circulating source for fibrinolysis, new antithrombotic messengers.

International audienceThrombus lysis is the consequence of a restricted number of reactions localised on the surface of fibrin and cell membranes. A functional defect or an insufficient fibrinolytic response may result in thrombosis with severe or fatal clinical consequences. Despite this clinical exigency and a real progress in the knowledge of the different components of this system (plasminogen activators, inhibitors and receptors) including structure-function relationship unveiled by the crystal structure of plasminogen, the functional evaluation of fibrinolysis still remains a challenge in haemostasis. Interestingly, we recently discovered that circulating membrane microvesicles might be indicators of the fibrinolytic response to an inflammatory or prothrombotic process via their participation in a new mechanism of plasmin formation requiring a cross-talk between two different surfaces. We propose that the fibrinolytic activity conveyed by microvesicles could be the real source of fibrinolysis in circulating blood

Nouveaux mécanismes fibrinolytiques des nanovésicules cellulaires (Détection et relevance physiopathologique)

PARIS7-Bibliothèque centrale (751132105) / SudocSudocFranceF

Fibrinolysis, new concepts: fibrinolytic microvesicles and cross-talk Fibrinolyse, nouveaux concepts : microvésicules et cross-talk fibrinolytiques

International audienceThrombus lysis is the consequence of a restricted number of reactions localised to the surface of fibrin. A functional defect or an insufficient fibrinolytic response may lead to thrombosis with severe or fatal clinical consequences, e.g. myocardial infarction and ischemic stroke. Despite this clinical exigency and a real progress in the knowledge of the different components of this system (plasminogen and its activators, inhibitors and receptors), its functional evaluation still remains a challenge in haemostasis. The absolute requirement of a template for molecular assembly of plasminogen and its activators (tissue- and urokinase-type plasminogen activators: tPA and uPA) restricts the formation of plasmin and protection of its activity to the surface of, respectively, fibrin and cells. In contrast, plasmin and tPA released from the clot during its lysis are immediately neutralised by their respective inhibitors α2-antiplasmin and plasminogen activator inhibitor 1, PAI-1). It seems therefore almost impossible to detect fibrinolytic activity in plasma with methods currently in use. Because of its unavailability, it is also impossible to measure the degree of fibrinolysis directly on the clot. Notwithstanding, it was recently discovered that circulating membrane microvesicles might be indicators of the fibrinolytic response to an inflammatory or prothrombotic process. These cell-derived fibrinolytic microvesicles bear at their membrane the plasminogen activators expressed by the parent cell: tPA from endothelial cells and uPA from leukocytes. These molecules are localised at the membrane surface and have the capacity to activate plasminogen into plasmin in situ. Moreover, it was recently discovered that these microvesicles might participate in a new mechanism of plasmin formation requiring a cross-talk between two different surfaces. In this fibrinolytic cross-talk one of the surfaces bear plasminogen (fibrin, extracellular matrix or platelets) whereas the other surface carry the plasminogen activator, typically leukocyte-derived microvesicles bearing uPA. These new actors and concepts in plasminogen activation represent hitherto unknown pathways in our comprehension of fibrinolysis and potential novel biomarkers in clinical practice