Jean Ripoche

Doppler resistive index to reflect regulation of renal vascular tone during sepsis and acute kidney injury

IntroductionRenal 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).MethodsThis 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.ResultsMedian 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.ConclusionsA 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.

Tissue inhibitors of matrix metalloproteinases in platelets and megakaryocytes: A novel organization for these secreted proteins

OBJECTIVE 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. MATERIALS AND METHODS 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. RESULTS 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. CONCLUSIONS TIMPs are synthesized and organized in MKs and platelets independently of other secreted proteins present in alpha-granule pools.

Flt3-ligand induces adhesion of haematopoietic progenitor cells via a very late antigen (VLA)-4- and VLA-5-dependent mechanism.

Summary. The adhesion of haematopoietic progenitor cells (HPC) to the bone marrow microenvironment is a process regulated by cytokines. In this study, we have shown that flt3‐ligand (FL), a growth factor that controls early haematopoiesis, regulated the function and expression of the beta‐1 integrins, very late antigen (VLA)‐4 and VLA‐5 on HPC. The modulation of the adhesiveness of HPC by FL was studied by adhesion assays on umbilical vein endothelial cells (HUVEC). Stimulation by FL induced two peaks of increased adhesiveness of HPC. The first peak was at around 30 min and was mechanistically related to an activation of the beta‐1 integrins, mainly VLA‐4 and VLA‐5. The second peak was at around 12 h and was related to increased expression of VLA‐4 and VLA‐5. The control of HPC adhesiveness by FL is a previously unreported property of FL that may be important for the homing and the retention of flt3‐expressing HPC within the bone marrow microenvironment.

Activation of the endothelium by IL-1α and glucocorticoids results in major increase of complement C3 and factor B production and generation of C3a

Constitutive secretion of complement C3 and factor B by the endothelial cell (EC) is lowered by therapeutic concentrations of glucocorticoids such as hydrocortisone or dexamethasone, whereas regulatory protein factor H production is increased by these hormones. In contrast, the pro‐inflammatory cytokine IL‐1α has a stimulatory effect on C3 and factor B secretion by the endothelium and an inhibitory effect on factor H secretion. In this study, we examined the combined effect of IL‐1α and glucocorticoids on C3 and factor B expression by the endothelial cell. When dexamethasone or hydrocortisone were added to IL‐1α, significant potentialization of IL‐1α‐induced stimulation of C3 and factor B production was observed, occurring at various concentrations of either stimuli. Dose‐response experiments indicate that, in vitro, optimal concentrations are in the range of 10−7 to 10−5m for dexamethasone and 50–200 U for IL‐1α. In contrast, dexamethasone counteracts, in an additive way, the inhibitory effect of IL‐1α on regulatory complement protein factor H production by EC. Such a potentialization between glucocorticoids and IL‐1α was not observed for another marker of endothelial activation, IL‐1α‐induced stimulation of coagulation tissue factor expression. The association of glucocorticoids and IL‐1α therefore appears to be a specific and major stimulus for the secretion of complement C3 and factor B, two acute‐phase proteins, by the endothelium. As a result of the in vitro endothelium stimulation by glucocorticoids and IL‐1α, C3a is generated in the vicinity of the endothelial cell. This study further suggests that complement activation, with its deleterious consequences, may result from the stimulation of endothelium in situations where high levels of IL‐1α and endogenous glucocorticoids coexist, such as in septic shock.

The transport of high amounts of vascular endothelial growth factor by blood platelets underlines their potential contribution in systemic sclerosis angiogenesis

OBJECTIVES 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. METHODS 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. RESULTS 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. CONCLUSIONS 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.

Blood platelets and inflammation: Their relationship with liver and digestive diseases

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.

Kinetic eGFR and Novel AKI Biomarkers to Predict Renal Recovery

BACKGROUND AND OBJECTIVES Prompt recognition of severe renal impairment could improve the early management of critically ill patients. We compared the value of kinetic eGFR, plasma neutrophil gelatinase-associated lipocalin (NGAL), and urine tissue inhibitor of metalloproteinase-2 and urine insulin-like growth factor-binding protein 7 ([TIMP-2]*[IGFBP7]) in predicting short-term recovery from AKI and major adverse kidney events. DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS During the 6-month study period, 245 patients were admitted to our intensive care unit. This study included 57 consecutive patients presenting with AKI within the first 24 hours after admission. AKI markers were evaluated at inclusion (day 0) and 24 hours later (day 1). Kinetic eGFR was calculated on day 1 according to serum creatinine evolution. Renal recovery was defined as normalization of serum creatinine with reversal of oliguria within 48 hours. Major adverse kidney events included death, need for RRT, or persistence of renal dysfunction at hospital discharge. RESULTS Plasma NGAL and [TIMP-2]*[IGFBP7] predicted renal recovery, with area under the receiver-operating characteristic curve (AUC-ROC) values between 0.70 and 0.79 at inclusion. Although plasma NGAL values frequently reached the maximal measurement range, their decrease on day 1 predicted recovery. The kinetic eGFR calculation after initial resuscitation provided the best AUC-ROC value for renal recovery, at 0.87. The best predictions for major adverse kidney events were provided by [TIMP-2]*[IGFBP7] and kinetic eGFR (equal AUC-ROCs of 0.81). Combining AKI markers in addition to clinical prediction models improved the discrimination and reclassification of patients who will recover from AKI or suffer from major adverse kidney events. CONCLUSIONS Biomarkers of kidney damage predicted short-term renal recovery and major adverse kidney events for an unselected cohort of critically ill patients. Calculating the kinetic eGFR imposed a delay after initial resuscitation but provided a good diagnostic and prognostic approach. The utility of functional and damage AKI marker combinations in addition to clinical information requires validation in larger prospective studies.

The ARE-associated factor AUF1 binds poly(A) in vitro in competition with PABP

The ARE (AU-rich element) is a post-transcriptional element controlling both mRNA turnover and translation initiation by primarily inducing poly(A) tail shortening. The mechanisms by which the ARE-associated proteins induce deadenylation are still obscure. One possibility among others would be that an ARE-ARE-BP (ARE-binding protein) complex intervenes in the PABP [poly(A)-binding protein]-poly(A) tail association and facilitates poly(A) tail accessibility to deadenylases. Here, we show by several experimental approaches that AUF1 (AU-rich element RNA-binding protein 1)/hnRNP (heterogeneous nuclear ribonucleoprotein) D, an mRNA-destabilizing ARE-BP, can bind poly(A) sequence in vitro. First, endogenous AUF1 proteins from HeLa cells specifically bound poly(A), independently of PABP. Secondly, using polyadenylated RNA probes, we showed that (i) the four recombinant AUF1 isoforms bind poly(A) as efficiently as PABP, (ii) the AUF1 binding to poly(A) does not change when the polyadenylated probe contains the GM-CSF (granulocyte/macrophage-colony stimulating factor) ARE, suggesting that, in vitro, the AUF1-poly(A) association was independent of the ARE sequence itself. In vitro, the binding of AUF1 isoforms to poly(A) displayed oligomeric and co-operative properties and AUF1 efficiently displaced PABP from the poly(A). Finally, the AUF1 molar concentration in HeLa cytoplasm was only 2-fold lower than that of PABP, whereas in the nucleus, its molar concentration was similar to that of PABP. These in vitro results suggest that, in vivo, AUF1 could compete with PABP for the binding to poly(A). Altogether, our results may suggest a role for AUF1 in controlling PABP-poly(A) tail association.

Immunohistochemical study of the phenotypic change of the mesenchymal cells during portal tract maturation in normal and fibrous (ductal plate malformation) fetal liver

BackgroundIn 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 Ivemarks syndrome.ResultsAt 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.ConclusionAs 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.

Iron particle labeling of haematopoietic progenitor cells: an in vitro study.

We present a method for labeling bone marrow haematopoietic progenitor cells with iron particles. Labeling was assessed by magnetic resonance imaging and electron microscopy. Labeling with iron particles could allow the following by imaging techniques of haematopoietic cells in physiologic and pathologic conditions such as the engraftment of haematopoietic progenitor cells or the migration of myelomonocytic cells in inflammatory diseases.