[Show abstract][Hide abstract] ABSTRACT: The role of platelets extends beyond hemostasis. The pivotal role of platelets in inflammation has shed new light on the natural history of conditions associated with acute or chronic inflammation. Beyond the preservation of vascular integrity, platelets are essential to tissue homeostasis and platelet-derived products are already used in the clinics. Unanticipated was the role of platelets in the adaptative immune response, allowing a renewed conceptual approach of auto-immune diseases. Platelets are also important players in cancer growth and dissemination. Platelets fulfill most of their functions through the expression of still incompletely characterized membrane-bound or soluble mediators. Among them, CD154 holds a peculiar position, as platelets represent a major source of CD154 and as CD154 contributes to most of these new platelet attributes. Here, we provide an overview of some of the new frontiers that the study of platelet CD154 is opening, in inflammation, tissue homeostasis, immune response, hematopoiesis and cancer.
[Show abstract][Hide abstract] ABSTRACT: Shear stress is one of mechanical constraints which are exerted by blood flow on endothelial cells (ECs). To adapt to shear stress, ECs align in the direction of flow through adherens junction (AJ) remodeling. However, mechanisms regulating ECs alignment under shear stress are poorly understood. The scaffold protein IQ domain GTPase activating protein 1 (IQGAP1) is a scaffold protein which couples cell signaling to the actin and microtubule cytoskeletons and is involved in cell migration and adhesion. IQGAP1 also plays a role in AJ organization in epithelial cells. In this study, we investigated the potential IQGAP1 involvement in the endothelial cells alignment under shear stress. Progenitor-derived endothelial cells (PDECs), transfected (or not) with IQGAP1 small interfering RNA, were exposed to a laminar shear stress (1.2 N/m(2)) and AJ proteins (VE-cadherin and β-catenin) and IQGAP1 were labeled by immunofluorescence. We show that IQGAP1 is essential for ECs alignment under shear stress. We studied the role of IQGAP1 in AJs remodeling of PDECs exposed to shear stress by studying cell localization and IQGAP1 interactions with VE-cadherin and β-catenin by immunofluorescence and Proximity Ligation Assays. In static conditions, IQGAP1 interacts with VE-cadherin but not with β-catenin at the cell membrane. Under shear stress, IQGAP1 lost its interaction from VE-cadherin to β-catenin. This "switch" was concomitant with the loss of β-catenin/VE-cadherin interaction at the cell membrane. This work shows that IQGAP1 is essential to ECs alignment under shear stress and that AJ remodeling represents one of the mechanisms involved. These results provide a new approach to understand ECs alignment under to shear stress.
PLoS ONE 11/2013; 8(11):e79919. DOI:10.1371/journal.pone.0079919 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Introduction
Renal resistive index (RI), determined by Doppler ultrasonography, directly reveals and quantifies modifications in renal vascular resistance. The aim of this study was to evaluate if mean arterial pressure (MAP) is determinant of renal RI in septic, critically ill patients suffering or not from acute kidney injury (AKI).
This prospective observational study included 96 patients. AKI was defined according to RIFLE criteria and transient or persistent AKI according to renal recovery within 3 days.
Median renal RIs were 0.72 (0.68-0.75) in patients without AKI and 0.76 (0.72-0.80) in patients with AKI (P=0.001). RIs were 0.75 (0.72-0.79) in transient AKI and 0.77 (0.70-0.80) in persistent AKI (P=0.84). RI did not differ in patients given norepinephrine infusion and was not correlated with norepinephrine dose. RI was correlated with MAP (ρ= -0.47; P=0.002), PaO2/FiO2 ratio (ρ= -0.33; P=0.04) and age (ρ=0.35; P=0.015) only in patients without AKI.
A poor correlation between renal RI and MAP, age, or PaO2/FiO2 ratio was found in septic and critically ill patients without AKI compared to patients with AKI. These findings suggest that determinants of RI are multiple. Renal circulatory response to sepsis estimated by Doppler ultrasonography cannot reliably be predicted simply from changes in systemic hemodynamics. As many factors influence its value, the interest in a single RI measurement at ICU admission to determine optimal MAP remains uncertain.
Critical care (London, England) 09/2012; 16(5):R165. DOI:10.1186/cc11517
[Show abstract][Hide abstract] ABSTRACT: IQGAP1 is a scaffold protein that interacts with proteins of the cytoskeleton and the intercellular adhesion complex. In podocytes, IQGAP1 is associated with nephrin in the glomerular slit diaphragm (SD) complex, but its role remains ill-defined. In this work, we investigated the interaction of IQGAP1 with the cytoskeleton and SD proteins in podocytes in culture, and its role in podocyte migration and permeability. Expression, localization, and interactions between IQGAP1 and SD or cytoskeletal proteins were determined in cultured human podocytes by Western blot (WB), immunocytolocalization (IC), immunoprecipitation (IP), and In situ Proximity Ligation assay (IsPL). Involvement of IQGAP1 in migration and permeability was also assessed. IQGAP1 expression in normal kidney biopsies was studied by immunohistochemistry. IQGAP1 expression by podocytes increased during their in vitro differentiation. IC, IP, and IsPL experiments showed colocalizations and/or interactions between IQGAP1 and SD proteins (nephrin, MAGI-1, CD2AP, NCK 1/2, podocin), podocalyxin, and cytoskeletal proteins (α-actinin-4). IQGAP1 silencing decreased podocyte migration and increased the permeability of a podocyte layer. Immunohistochemistry on normal human kidney confirmed IQGAP1 expression in podocytes and distal tubular epithelial cells and also showed an expression in glomerular parietal epithelial cells. In summary, our results suggest that IQGAP1, through its interaction with components of SD and cytoskeletal proteins, is involved in podocyte barrier properties.
PLoS ONE 05/2012; 7(5):e37695. DOI:10.1371/journal.pone.0037695 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: An expansion of knowledge from basic and clinical research has highlighted the critical role of platelets in inflammation and tissue repair in addition to their established contribution to hemostasis. Activated platelets are a rich source of mediators participating to inflammation and tissue regeneration. Platelet-derived microparticles recapitulate essential platelet functions and their contribution to the pathogenesis of inflammatory diseases has been emphasized. Recent findings suggest that platelets are both friends and foes for the liver. Platelets are essential to liver regeneration, platelet-derived serotonin being critical. However platelets can also exacerbate liver damage, as in immune-mediated injury. The dual role of platelets has recently been exemplified in animal models of liver fibrosis. Platelets release profibrogenic mediators, such as CXC Chemokine Ligand 4, that is instrumental in the progression of liver fibrosis. On the other hand, thrombocytopenia aggravates liver fibrosis, an outcome linked to the downregulation of hepatic stellate cell collagen production by platelet derived hepatocyte growth factor. CD154, a key molecule in inflammation, is expressed by platelets and is a pathogenic mediator in inflammatory bowel disease. Here, we summarize some of the mechanisms linking platelets with inflammation and comment few recent articles indicating why platelets may prove to be important pathogenic mediators in liver and gastrointestinal diseases.
[Show abstract][Hide abstract] ABSTRACT: Inflammation and lipid metabolism pathways are linked, and deregulation of this interface may be critical in hepatic steatosis. The importance of the dialog between inflammatory signaling pathways and the unfolded protein response (UPR) in metabolism has been underlined. Herein, we studied the role of CD154, a key mediator of inflammation, in hepatic steatosis. To this end, Balb/c mice, wild-type or deficient in CD154 (CD154KO), were fed a diet rich in olive oil. In vitro, the effect of CD154 was studied on primary hepatocyte cultures and hepatocyte-derived cell lines. Results showed that CD154KO mice fed a diet rich in olive oil developed hepatic steatosis associated with reduced apolipoprotein B100 (apoB100) expression and decreased secretion of very low-density lipoproteins. This phenotype correlated with an altered UPR as assessed by reduced X-Box binding protein-1 (XBP1) messenger RNA (mRNA) splicing and reduced phosphorylation of eukaryotic initiation factor 2α. Altered UPR signaling in livers of CD154KO mice was confirmed in tunicamycin (TM) challenge experiments. Treatment of primary hepatocyte cultures and hepatocyte-derived cell lines with soluble CD154 increased XBP1 mRNA splicing in cells subjected to either oleic acid (OA) or TM treatment. Moreover, CD154 reduced the inhibition of apoB100 secretion by HepG2 cells grown in the presence of high concentrations of OA, an effect suppressed by XBP1 mRNA silencing and in HepG2 cells expressing a dominant negative form of inositol requiring ER-to-nucleus signaling protein-1. The control of the UPR by CD154 may represent one of the mechanisms involved in the pathophysiology of hepatic steatosis. Conclusion: Our study identifies CD154 as a new mediator of hepatic steatosis.
[Show abstract][Hide abstract] ABSTRACT: Elastic fibers are composed of microfibrils containing fibrillin-1 and an elastic component, elastin. Microfibrils may not be associated with elastin. In the adult liver, fibrillin-1 and elastin are coexpressed within the stroma and portal tracts vessel walls. Fibrillin-1 is expressed alone around the bile ducts and within the Disse space. There is little work that has studied the elastic fiber organization during the fœtal liver development. Here, we studied the expression of fibrillin-1 and elastin by immunohistochemistry on 20 cases of fœtal liver. During the development of the portal tract, the two components are coexpressed on interstitial elastic fibers and within vessel walls. Fibrillin-1 is expressed alone around the bile structures during their maturation. Unlike adult liver, fibrillin-1 is expressed on thin and very irregular microfibrils within the Disse space. Our study shows that the elastic matrix development in the portal tract follows the development of the different structures, notably biliary structures. In the Disse space, microfibrils are not continuous. Their maturation may be in relation with the change of the hepatic blood flow after birth.
[Show abstract][Hide abstract] ABSTRACT: Altered angiogenesis is a characteristic feature in SSc and remains ill-understood. VEGF is believed to play a central role. Serum VEGF is elevated in SSc patients but questions remain concerning the source of circulating VEGF. Here we investigated platelet activation and the role of platelets as a source of VEGF and other angiogenic mediators in this disease.
A cohort of 40 patients with SSc was included. Age- and sex-matched healthy subjects and subjects presenting a primary RP were included as controls. Platelets were isolated, activated with thrombin and the secretion of VEGF, platelet derived growth factor, homodimeric form BB (PDGF-BB), TGF-beta1 and angiopoietins-1 and -2 measured. Plasma concentrations of these mediators and the functionality of platelet-derived VEGF were also studied. Platelet activation was assayed by measuring plasma beta-thromboglobulin and expression of P-selectin on platelets. The effect of iloprost on VEGF secretion by platelets was studied.
Platelets from SSc patients, in contrast to controls, secreted large amounts of VEGF when activated, but not PDGF-BB, TGF-beta1 or angiopoietins. Increased expression of membrane P-selectin confirmed platelet activation in the patients. Iloprost inhibited VEGF secretion by platelets both in vivo and in vitro, through inhibition of platelet activation.
Platelets transport high levels of VEGF in SSc. They may contribute to circulating VEGF because of ongoing activation in the course of the disease. If activated at the contact of injured endothelium, platelets may be important in the altered angiogenesis associated with the disease through the secretion of high levels of VEGF.
[Show abstract][Hide abstract] ABSTRACT: Expression of tissue inhibitors of matrix metalloproteinases (TIMPs) is one way that activated platelets intervene in tissue remodeling and angiogenesis. Our study was designed to investigate their synthesis in megakaryocytes (MKs) and their storage in platelets.
TIMP expression in MKs derived from blood CD34(+) progenitor cells of normal donors and a megakaryocytic cell line (CHRF-288-11) grown in serum-free conditions and platelets from normal donors or two patients with gray platelet syndrome was studied by immunofluorescence labeling, reverse transcription-polymerase chain reaction, and western blotting.
Biosynthesis of TIMPs 1-4 in MKs was indicated by presence of their messenger RNAs as shown by polymerase chain reaction and of their proteins. Immunofluorescence labeling suggested a primarily granular localization of TIMPs in MKs and platelets. But when colocalization with von Willebrand factor, fibrinogen, P-selectin, and other alpha-granule proteins was assessed in platelets by confocal microscopy, TIMP-1, -2, and -4 were localized as distinct fluorescent patches apart from the established alpha-granule markers and largely independent of platelet metalloproteinases. TIMP-3 differed for it also had an alpha-granule location. Western blotting confirmed the presence of TIMPs 1-4 in platelets and thrombin activation resulted in their extensive release to the medium. Platelets from two patients with gray platelet syndrome, congenitally deficient in alpha-granules, showed sparse labeling of von Willebrand factor and fibrinogen confined to vestigial alpha-granules; however, localization of the TIMPs was unchanged.
TIMPs are synthesized and organized in MKs and platelets independently of other secreted proteins present in alpha-granule pools.
[Show abstract][Hide abstract] ABSTRACT: In adult liver, the mesenchymal cells, portal fibroblasts and vascular smooth muscle cells can transdifferentiate into myofibroblasts, and are involved in portal fibrosis. Differential expression of markers, such as alpha-smooth muscle actin (ASMA), h-caldesmon and cellular retinol-binding protein-1 allows their phenotypic discrimination. The aim of our study was to explore the phenotypic evolution of the mesenchymal cells during fetal development in normal liver and in liver with portal fibrosis secondary to ductal plate malformation in a series of Meckel-Gruber syndrome, autosomal recessive polycystic kidney disease and Ivemark's syndrome.
At the early steps of the portal tract maturation, portal mesenchymal cells expressed only ASMA. During the maturation process, these cells were found condensed around the biliary and vascular structures. At the end of maturation process, only cells around vessels expressed ASMA and cells of the artery tunica media also expressed h-caldesmon. In contrast, ASMA positive cells persisted around the abnormal biliary ducts in fibrous livers.
As in adult liver, there is a phenotypic heterogeneity of the mesenchymal cells during fetal liver development. During portal tract maturation, myofibroblastic cells disappear in normal development but persist in fibrosis following ductal plate malformation.