[Amiodarone-induced optic neuropathy: A rare side effect]. / Un effet indésirable rare de l'amiodarone : la neuropathie optique.

INTRODUCTION: The diagnosis of bilateral papilledema implies emergency medical care to look for intracranial hypertension and arteritic ischemic neuropathy. However, other causes must also be mentioned, including drugs. Too often underrated because of their usual benignity, drug side ophthalmological effects can be severe and are typically bilateral. CASE REPORT: An 80-year-old woman was hospitalized for bilateral papilledema, predominantly in the left eye, with lowered visual acuity. After ruling out intracranial hypertension, arteritic ischemic optic neuropathy, non-arteritic, and inflammatory bilateral papilledema, the diagnosis was toxic optic neuropathy. CONCLUSION: Bilateral edematous optic neuropathy is a known side effect of amiodarone, uncommon but to be known because of the large number of patients benefiting from this treatment.

Development of a strategy for the quantification of food allergens in several food products by mass spectrometry in a routine laboratory.

Worldwide, mass spectrometry is widely used to detect and quantify food allergens, especially in complex and processed food products. Yet, the absence of a regulatory framework for the developed methods has led to a lack of harmonization between laboratories. In this study, ten allergens were analyzed in eight food products by UHPLC-MS/MS, in order to establish criteria for the retention time, variation tolerance, the ion ratio deviation, and the signal-to-noise ratio for allergen detection. The set of criteria should help laboratories to compare results and avoid false positives and negatives. Furthermore, a strategy combining standard addition and labeled peptide correction was used to quantify milk, soy, peanut, and egg allergens in eight food products. This strategy is particularly interesting for routine laboratories, which receive hundreds of samples and cannot use an external calibration curve for each sample.

Hypoxia counteracts taxol-induced apoptosis in MDA-MB-231 breast cancer cells: role of autophagy and JNK activation.

Cancer cell resistance against chemotherapy is still a heavy burden to improve anticancer treatments. Autophagy activation and the development of hypoxic regions within the tumors are known to promote cancer cell resistance. Therefore, we sought to evaluate the role of autophagy and hypoxia on the taxol-induced apoptosis in MDA-MB-231 breast cancer cells. The results showed that taxol induced apoptosis after 16 h of incubation, and that hypoxia protected MDA-MB-231 cells from taxol-induced apoptosis. In parallel, taxol induced autophagy activation already after 2 h of incubation both under normoxia and hypoxia. Autophagy activation after taxol exposure was shown to be a protective mechanism against taxol-induced cell death both under normoxia and hypoxia. However, at longer incubation time, the autophagic process reached a saturation point under normoxia leading to cell death, whereas under hypoxia, autophagy flow still correctly took place allowing the cells to survive. Autophagy induction is induced after taxol exposure via mechanistic target of rapamycin (mTOR) inhibition, which is more important in cells exposed to hypoxia. Taxol also induced c-Jun N-terminal kinase (JNK) activation and phosphorylation of its substrates B-cell CLL/lymphoma 2 (Bcl2) and BCL2-like 1 (BclXL) under normoxia and hypoxia very early after taxol exposure. Bcl2 and BclXL phosphorylation was decreased more importantly under hypoxia after long incubation time. The role of JNK in autophagy and apoptosis induction was studied using siRNAs. The results showed that JNK activation promotes resistance against taxol-induced apoptosis under normoxia and hypoxia without being involved in induction of autophagy. In conclusion, the resistance against taxol-induced cell death observed under hypoxia can be explained by a more effective autophagic flow activated via the classical mTOR pathway and by a mechanism involving JNK, which could be dependent on Bcl2 and BclXL phosphorylation but independent of JNK-induced autophagy activation.

Unbiased proteomic analysis of proteins interacting with the HIV-1 5'LTR sequence: role of the transcription factor Meis.

To depict the largest picture of a core promoter interactome, we developed a one-step DNA-affinity capture method coupled with an improved mass spectrometry analysis process focused on the identification of low abundance proteins. As a proof of concept, this method was developed through the analysis of 230 bp contained in the 5'long terminal repeat (LTR) of the human immunodeficiency virus 1 (HIV-1). Beside many expected interactions, many new transcriptional regulators were identified, either transcription factors (TFs) or co-regulators, which interact directly or indirectly with the HIV-1 5'LTR. Among them, the homeodomain-containing TF myeloid ectopic viral integration site was confirmed to functionally interact with a specific binding site in the HIV-1 5'LTR and to act as a transcriptional repressor, probably through recruitment of the repressive Sin3A complex. This powerful and validated DNA-affinity approach could also be used as an efficient screening tool to identify a large set of proteins that physically interact, directly or indirectly, with a DNA sequence of interest. Combined with an in silico analysis of the DNA sequence of interest, this approach provides a powerful approach to select the interacting candidates to validate functionally by classical approaches.

mtCLIC is up-regulated and maintains a mitochondrial membrane potential in mtDNA-depleted L929 cells.

To explain why mitochondrial DNA (mtDNA)-depleted or rho0 cells still keep a mitochondrial membrane potential (Delta(psi)m) in the absence of respiration, several hypotheses have been proposed. The principal and well accepted one involves a reverse of action for ANT combined to F1-ATPase activity. However, the existence of other putative electrogenic channels has been speculated. Here, using mRNA differential display reverse transcriptase-polymerase chain reaction on L929 mtDNA-depleted cells, we identified mtCLIC as a differentially expressed gene in cells deprived from mitochondrial ATP production. Mitochondrial chloride intracellular channel (mtCLIC), a member of a recently discovered and expanding family of chloride intracellular channels, is up-regulated in mtDNA-depleted and rho0 cells. We showed that its expression is dependent on CREB and p53 and is sensitive to calcium and tumor necrosis factor alpha. Interestingly, up- or down-regulation of mtCLIC protein expression changes Delta(psi)m whereas the chloride channel inhibitor NPPB reduces the Delta(psi)m in mtDNA-depleted L929 cells, measured with the fluorescent probe rhodamine 123. Finally, we demonstrated that purified mitochondria from mtDNA-depleted cells incorporate, in a NPPB-sensitive manner, more 36chloride than parental mitochondria. These findings suggest that mtCLIC could be involved in mitochondrial membrane potential generation in mtDNA-depleted cells, a feature required to prevent apoptosis and to drive continuous protein import into mitochondria.

CREB activation induced by mitochondrial dysfunction is a new signaling pathway that impairs cell proliferation.

We characterized a new signaling pathway leading to the activation of cAMP-responsive element-binding protein (CREB) in several cell lines affected by mitochondrial dysfunction. In vitro kinase assays, inhibitors of several kinase pathways and overexpression of a dominant-negative mutant for calcium/calmodulin kinase IV (CaMKIV), which blocks the activation of CREB, showed that CaMKIV is activated by a mitochondrial activity impairment. A high calcium concentration leading to the disruption of the protein interaction with protein phosphatase 2A explains CaMKIV activation in these conditions. Transcrip tionally active phosphorylated CREB was also found in a rho0 143B human osteosarcoma cell line and in a MERRF cybrid cell line mutated for tRNA(Lys) (A8344G). We also showed that phosphorylated CREB is involved in the proliferation defect induced by a mitochondrial dysfunction. Indeed, cell proliferation inhibition can be prevented by CaMKIV inhibition and CREB dominant-negative mutants. Finally, our data suggest that phosphorylated CREB recruits p53 tumor suppressor protein, modifies its transcriptional activity and increases the expression of p21(Waf1/Cip1), a p53-regulated cyclin-dependent kinase inhibitor.

Hypoxia-induced increase in intracellular calcium concentration in endothelial cells: role of the Na(+)-glucose cotransporter.

Hypoxia is a common denominator of many vascular disorders, especially those associated with ischemia. To study the effect of oxygen depletion on endothelium, we developed an in vitro model of hypoxia on human umbilical vein endothelial cells (HUVEC). Hypoxia strongly activates HUVEC, which then synthesize large amounts of prostaglandins and platelet-activating factor. The first step of this activation is a decrease in ATP content of the cells, followed by an increase in the cytosolic calcium concentration ([Ca(2+)](i)) which then activates the phospholipase A(2) (PLA(2)). The link between the decrease in ATP and the increase in [Ca(2+)](i) was not known and is investigated in this work. We first showed that the presence of extracellular Na(+) was necessary to observe the hypoxia-induced increase in [Ca(2+)](i) and the activation of PLA(2). This increase was not due to the release of Ca(2+) from intracellular stores, since thapsigargin did not inhibit this process. The Na(+)/Ca(2+) exchanger was involved since dichlorobenzamil inhibited the [Ca(2+)](i) and the PLA(2) activation. The glycolysis was activated, but the intracellular pH (pH(i)) in hypoxic cells did not differ from control cells. Finally, the hypoxia-induced increase in [Ca(2+)](i) and PLA(2) activation were inhibited by phlorizin, an inhibitor of the Na(+)-glucose cotransport. The proposed biochemical mechanism occurring under hypoxia is the following: glycolysis is first activated due to a requirement for ATP, leading to an influx of Na(+) through the activated Na(+)-glucose cotransport followed by the activation of the Na(+)/Ca(2+) exchanger, resulting in a net influx of Ca(2+).

Endothelial cell responses to hypoxia: initiation of a cascade of cellular interactions.

The origin of several vascular pathologies involves sudden or recurrent oxygen deficiency. In this review, we examine what the biochemical and molecular responses of the endothelial cells to the lack of oxygen are and how these responses may account for the features observed in pathological situations, mainly by modifications of cell-cell interactions. Two major responses of the endothelial cells have been observed depending on the degree and duration of the oxygen deficiency. Firstly, acute hypoxia rapidly activates the endothelial cells to release inflammatory mediators and growth factors. These inflammatory mediators are able to recruit and promote the adherence of neutrophils to the endothelium where they become activated. The synthesis of platelet-activating factor plays a key role in this adherence process. Secondly, longer periods of hypoxia increase the expression of specific genes such as those encoding some cytokines as well as for the growth factors platelet-derived growth factor and vascular endothelial growth factor. The transcriptional induction of these genes is mediated through the activation of several transcription factors, the most important one being hypoxia inducible factor-1. The link between our knowledge of the signalling cascade of the cellular and molecular events initiated by hypoxia and their involvement in several vascular pathological situations, varicose veins, tumor angiogenesis and pulmonary hypertension is discussed briefly.

Interaction between RGS7 and polycystin.

Regulators of G protein signaling (RGS) proteins accelerate the intrinsic GTPase activity of certain Galpha subunits and thereby modulate a number of G protein-dependent signaling cascades. Currently, little is known about the regulation of RGS proteins themselves. We identified a short-lived RGS protein, RGS7, that is rapidly degraded through the proteasome pathway. The degradation of RGS7 is inhibited by interaction with a C-terminal domain of polycystin, the protein encoded by PKD1, a gene involved in autosomal-dominant polycystic kidney disease. Furthermore, membranous expression of C-terminal polycystin relocalized RGS7. Our results indicate that rapid degradation and interaction with integral membrane proteins are potential means of regulating RGS proteins.

Cellular activation triggered by the autosomal dominant polycystic kidney disease gene product PKD2.

Autosomal dominant polycystic kidney disease (ADPKD) is caused by germ line mutations in at least three ADPKD genes. Two recently isolated ADPKD genes, PKD1 and PKD2, encode integral membrane proteins of unknown function. We found that PKD2 upregulated AP-1-dependent transcription in human embryonic kidney 293T cells. The PKD2-mediated AP-1 activity was dependent upon activation of the mitogen-activated protein kinases p38 and JNK1 and protein kinase C (PKC) epsilon, a calcium-independent PKC isozyme. Staurosporine, but not the calcium chelator BAPTA [1,2-bis(o-aminophenoxy)ethane-N,N,N', N'-tetraacetate], inhibited PKD2-mediated signaling, consistent with the involvement of a calcium-independent PKC isozyme. Coexpression of PKD2 with the interacting C terminus of PKD1 dramatically augmented PKD2-mediated AP-1 activation. The synergistic signaling between PKD1 and PKD2 involved the activation of two distinct PKC isozymes, PKC alpha and PKC epsilon, respectively. Our findings are consistent with others that support a functional connection between PKD1 and PKD2 involving multiple signaling pathways that converge to induce AP-1 activity, a transcription factor that regulates different cellular programs such as proliferation, differentiation, and apoptosis. Activation of these signaling cascades may promote the full maturation of developing tubular epithelial cells, while inactivation of these signaling cascades may impair terminal differentiation and facilitate the development of renal tubular cysts.

The polycystic kidney disease 1 gene product modulates Wnt signaling.

Two distinct signaling pathways, involving Wnt signaling and polycystin, have been found to be critical for normal kidney development. Renal tubulogenesis requires the presence of certain Wnt proteins, whereas mutations in polycystin impede the terminal differentiation of renal tubular epithelial cells, causing the development of large cystic kidneys that characterize autosomal dominant polycystic kidney disease. Polycystin is an integral membrane protein, consisting of several extracellular motifs indicative of cell-cell and cell-matrix interactions, coupled through multiple transmembrane domains to a functionally active cytoplasmic domain. We report here that expression of the C-terminal cytoplasmic domain of polycystin stabilizes soluble endogenous beta-catenin and stimulates TCF-dependent gene transcription in human embryonic kidney cells. Microinjection of the polycystin C-terminal cytoplasmic domain induces dorsalization in zebrafish. Our findings suggest that polycystin has the capacity to modulate Wnt signaling during renal development.

Effect of hydroxyethylrutosides on hypoxial-induced neutrophil adherence to umbilical vein endothelium.

A clinically available mixture of hydroxyethylrutosides (HR) was examined as a protector against endothelial cell activation by hypoxia in perfused human umbilical vein. The results showed that 500 micrograms/mL HR totally inhibited the adherence of human unstimulated neutrophils to the endothelium of umbilical vein incubated in hypoxic conditions. This inhibition was confirmed by a morphological study performed by scanning electron microscopy. In addition, neutrophils adherent to the hypoxic umbilical vein endothelium became activated, as evidence by the increased release of superoxide anions and synthesis of leukotriene B4. These processes could also be inhibited by HR. In conclusion, the results of this study suggest that the improvement in venous insufficiency observed clinically with HR could, in part, be the result of their ability to inhibit the recruitment and activation of neutrophils by endothelium activated during blood stasis.

Role of PECAM-1 in the adherence of PMN to hypoxic endothelial cells.

Hypoxia induces an increase in PMN adherence to endothelial cells for which an interaction between ICAM-1 and CD18/CD11b has been demonstrated. Since PECAM-1 has been shown to be involved in PMN transmigration through the endothelium and to increase the binding capacity of leukocyte CD18/CD11b, the role of this molecule in the hypoxia-induced PMN adherence was investigated. Hypoxia did not change the total surface expression of PECAM-1 on HUVEC and did not change the cell-cell border localization of this molecule as TNF-alpha did. In addition, blocking anti-PECAM-1 antibodies could not inhibit the increased adherence of unstimulated human PMN to hypoxia-incubated HUVEC while anti-ICAM-1 partially inhibited this process. These results indicate that PECAM-1 is probably not involved in the hypoxia-induced PMN adherence to endothelial cells.

Effect of Ginkor Fort on hypoxia-induced neutrophil adherence to human saphenous vein endothelium.

This study was performed to evaluate the effects of Ginkor Fort, a venotropic drug composed of Ginkgo biloba extract, troxerutine, and heptaminol, on neutrophil adherence to the endothelium of saphenous veins. When saphenous veins were incubated 2 h in hypoxic conditions, they showed a five- to sixfold increase in neutrophil adherence to the endothelium. Ginkor Fort at 0.3 mg/ml was able to inhibit this increase by 69%. These results were confirmed by observations in scanning electron microscopy. Ginkor Fort also inhibited the subsequent activation of these neutrophils, as evidenced by the inhibition of superoxide anion release. The biochemical mechanism of this inhibition of neutrophil adherence was studied on endothelial cells in culture. We observed that Ginkor Fort was able to inhibit the different steps of the activation of endothelial cells by hypoxia: the activation of phospholipase A2 and the decrease in adenosine triphosphate (ATP) content. By preventing the first step of the activation cascade, the decrease in ATP content, Ginkor Fort blocks the subsequent increase in neutrophil adherence as well as neutrophil activation. The biochemical mechanism evidenced in this work might explain the beneficial effect of this drug in the treatment of patients with chronic venous insufficiency.

Effect of aescine on hypoxia-induced activation of human endothelial cells.

Phlebotonic drugs are very often old drugs which improve symptoms in chronic venous insufficiency but their precise mechanism remains unclear. One reason for this lack of information is our poor understanding of the aetiology of the varicose vein. One hypothesis which is being more and more substantiated is that the origin of the disease lies in the activation of the endothelium during blood stasis, leading to a cascade of reactions which, in the long term, alter the structure of the vein wall. In this work, we tested aescine (Reparil i.v. form), a phlebotonic drug, in an in vitro model which mimics this situation, i.e. human endothelial cells exposed to hypoxic conditions. Aescine was shown to inhibit 2 important steps of the activation of endothelial cells incubated 120 min under hypoxia the decrease in ATP content, which is the starting point of the activation cascade, and the increase in the activity of phospholipase A2, an enzyme responsible for the release of precursors of inflammatory mediators. Hypoxia-activated endothelial cells also increase their adhesiveness for neutrophils. This process could also be prevented in a dose-dependent manner if endothelial cells were incubated in the presence of aescine. This inhibition was confirmed by morphological observations in scanning electron microscopy. All 3 effects were already evidenced at 100 ng/ml and were maximal at 750 ng/ml. These effects obtained at very low concentrations probably represent one of the main molecular and cellular mechanisms that underlie, among others, protection of the vessel wall. Objective criteria for our understanding of the preventive action of this phlebotonic drug are, thus, provided.

Effects of hydroxyethylrutosides on hypoxia-induced activation of human endothelial cells in vitro.

1. A clinically available mixture of hydroxyethylrutosides (HR) was examined as inhibitors of endothelial cell activation by hypoxia in vitro. Thus, the effects of HR on ATP depletion, phospholipase A2 activation and neutrophil adherence were investigated in hypoxia-activated human umbilical vein endothelial cells in primary cell culture. 2. Our results show that HR inhibited two important steps of the activation of endothelial cells by hypoxia: the decrease in ATP content, which is the starting point of the process, and the activation of phospholipase A2 one enzyme responsible for the release of inflammatory mediators. This inhibition was dose-dependent with 70 to 90% inhibition at 500 micrograms ml-1 of HR. 3. In addition, hypoxia-activated endothelial cells increased their adhesiveness for neutrophils. This process could also be prevented in a dose-dependent manner if endothelial cells were incubated in the presence of HR. This inhibition was confirmed by a morphological study. 4. In conclusion, the results of this study suggest that a possible explanation for the improvement in venous insufficiency by HR observed clinically could be their ability to inhibit the activation of endothelial cells during blood stasis.

Effects of naftidrofuryl on hypoxia-induced activation and mortality of human endothelial cells.

The present study was designed to elucidate the possible beneficial effects of naftidrofuryl on ischemia-induced endothelium damage. For this purpose, an in vitro model was developed wherein human endothelial cells isolated from umbilical vein were submitted to hypoxia. Long-term hypoxia incubation (6 h) induced cell mortality, and naftidrofuryl strongly protected endothelial cells against this mortality in a dose-dependent manner and at concentrations as low as 10(-9) M. 66% protection was still observed after 16 h of hypoxia. Naftidrofuryl had to be present during the hypoxia incubation to exert its action; preincubation up to 24 h in the presence of naftidrofuryl could not protect endothelial cells incubated under hypoxia without naftidrofuryl. Short-term hypoxia, which does not induce mortality, strongly activates the endothelial cells with an increase in the cytosolic calcium concentration, in the phospholipase A2 activity, and in the synthesis of prostaglandin and of platelet-activating factor. It also enhances the adherence of polymorphonuclear neutrophils. Naftidrofuryl was able to markedly inhibit this whole cascade of events in a dose-dependent manner. We also demonstrated that naftidrofuryl could block the decrease in ATP concentration that results from the hypoxic conditions. These results indicate that by preserving the energetic level of the cells, naftidrofuryl prevents the activation of endothelial cells and the cell mortality induced by hypoxia. By maintaining an intact endothelium in vivo during ischemia, naftidrofuryl could prevent the further damage induced by leukocyte recruitment and activation.

Hypoxia-induced activation of endothelial cells as a possible cause of venous diseases: hypothesis.

Blood stasis in leg veins is a situation commonly linked to the development of venous diseases such as varicoses. Such a stasis will provoke an ischemia, thus decreasing oxygen availability to tissues. Owing to its localization between blood and tissue, endothelium is the first target of this insult. The authors develop here a hypothesis in which the effect of oxygen deprivation on the functional state of the endothelium is the starting point of a cascade of events leading to the disorganization of the vessel wall typical of these pathologies. When venous human endothelial cells obtained from umbilical cords (HUVEC) are exposed to hypoxic conditions they become activated without change in their viability. The synthesis of a proinflammatory molecule (PAF, platelet-activating factor) and the adhesion of human polymorphonuclear neutrophils (PMN) on HUVEC are markedly increased during hypoxia incubation. These two processes are related to a calcium-dependent activation of endothelial cells due to a decrease of adenosine triphosphate (ATP) availability during hypoxia. Adherence of neutrophils to endothelial cells is the first step of diapedesis, which leads to the infiltration of these cells in the media of the veins, where they affect the smooth muscle cells and the connective tissue, leading to tissue alterations typical of the venous pathologies. The authors propose that this sequential process which originates from a reduction in oxygen availability and which involves different cell type as one main cause of the venous disorders, in addition to genetic, hormonal, and mechanical factors.

Effect of hypoxia upon intracellular calcium concentration of human endothelial cells.

Ischemia is a situation occurring in several diseases including myocardial infarction and organ transplantation in which oxygenated blood supply is impaired. Ischemia leads to many cellular and tissue modifications, the most important one being cell death. Several explanations have been proposed to account for these modifications and cell death; among them is calcium overload. However, the influence of calcium concentration on the alteration of endothelial cell functions or viability during ischemia are still unknown. We developed here an in vitro model where human endothelial cell monolayers were submitted to hypoxia with or without reoxygenation and variation in calcium concentration was followed using a specific intracellular probe Fura 2. We observed a significant increase of [Ca2+]i during 2 h hypoxia reaching values similar to those observed during agonist stimulation of endothelial cells but far lower than values toxic for the cells. This increase was constant during the hypoxic incubation and was due mainly to an influx of extracellular calcium. Viability was also followed during hypoxia and using calcium channel blockers, we could show that there was no correlation between viability and the rise in calcium concentration. During the reoxygenation period, [Ca2+]i decreased to reach the normal value of resting cells after 45 min, suggesting that cells were still able to recover their calcium homeostasis. The use of a ketone body (beta-hydroxybutyrate) indicated that an energy deficiency was responsible for the hypoxia-induced increase in [Ca2+]i. We actually observed a 43% decrease in ATP concentration after 2 h hypoxia. This decrease was already significant after 30 min which thus precedes the changes in [Ca2+]i. These results show that during hypoxia, energy deficiency led to an increase in [Ca2+]i which is, however, too low to account for the loss of viability but which is within the range of concentrations observed during stimulation of endothelial cells. We propose that such increased intracellular calcium concentrations could play a role in the synthesis of mediators leading to the development of local inflammation.