Biomedical subjects

T Arnould

Publications and source records attributed to T Arnould.

34 records · Page 2Linked to original sources

PubMed1997

Perfused human saphenous veins for the study of the origin of varicose veins: role of the endothelium and of hypoxia.

If venous stasis due to blood stagnation has been recognized to be involved in the development of varicose veins, the mechanism linking this situation to the modifications of the venous wall observed in varicoses is still unclear. In order to study this mechanism, human saphenous veins were incubated in normoxic or hypoxic conditions and the interactions between the endothelium and neutrophils were investigated. We observed that many neutrophils adhered to the endothelium of veins incubated in hypoxic conditions rather than in normoxia and that these adherent neutrophils were activated: they released high amounts of superoxide anion and of leukotriene B4. Studies in scanning electron microscopy confirmed the increased neutrophil adherence to the endothelium as well as their activation. These results were then related to the histological observation of varicose veins. These veins show a thickening of the media with extracellular matrix deposit as well as an alteration of the elastic lamina with the presence of smooth muscle cells in the intima. These results are in agreement with in vitro studies on isolated endothelial cells. They all show that hypoxia is able to activate endothelial cells: they release inflammatory mediators and become adhesive for neutrophils which are then activated. These activated leukocytes release free radicals and proteases which are able to degrade the extracellular matrix. In addition, hypoxia-activated endothelial cells secrete growth factors which will trigger smooth muscle cell proliferation and the synthesis of extracellular matrix components. Altogether and because they are frequently repeated, these processes could eventually lead to alterations of the venous wall similar to those observed in varicose veins.

Cell Adhesion↗

PubMed1996

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.

Adenosine Triphosphate↗

PubMed1996

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.

Adenosine Triphosphate↗

PubMed1996

Interactions between endothelial cells and smooth muscle cells after their activation by hypoxia. A possible etiology for venous disease.

Because of their localization at the interface between blood and tissue, endothelial cells are responsible for the maintenance of vascular homeostasis. They fulfil a series of various functions and constantly interact with circulating leukocytes and with the smooth muscle cells (SMC) present in the media. Any disturbance of their metabolism can thus lead to alterations of the blood vessel functions. We have shown that hypoxia, for example resulting from venous stasis, induces the activation of endothelial cells which then release inflammatory mediators able to activate neutrophils and to induce their infiltration as well as growth factors for SMC. We propose that these processes are the beginning of a cascade of events eventually leading to structural and functional modifications of the venous wall similar to the ones observed in varicose vein wall. The endothelium alterations resulting from venous stasis would thus be the origin of the development of the venous disease. Pharmacological and clinical evidence reinforce this hypothesis.

Cell Hypoxia↗

PubMed1995

Hypoxia induces PMN adherence to umbilical vein endothelium.

OBJECTIVE: In vitro incubation of cultured endothelial cells under hypoxia leads to the activation of these cells and results in an increase of their adhesiveness for neutrophils (PMN). Because of the possible relevance of these observations for pathological situations, we investigated whether adherence of PMN also occurs in an entire vein after its incubation in hypoxic conditions. METHODS: Human umbilical veins in complete cords were incubated for 2 h in normoxic or hypoxic conditions and the adherence of unstimulated human 51Cr-labelled-PMN was measured under flow conditions. Experiments with human umbilical vein endothelial cells (HUVEC) were performed in parallel for comparison. Morphological studies in scanning electron microscopy were carried out in both in vitro and ex vivo situations. RESULTS: Hypoxia induced an increase in the adherence of PMN either to HUVEC or to the umbilical vein endothelium up to 5- to 6-fold when compared to normoxic conditions (P < 0.001). In both cases, this hypoxia-induced adherence was inhibited by anti-ICAM-1 antibodies or when the PAF (platelet-activating factor) synthesis was blocked during hypoxia by oleic acid. Furthermore, the adherence of PMN was inhibited when PMN were pre-incubated with WEB 2086 (a selective PAF receptor antagonist). These results indicate a crucial role of PAF in this process. Morphological studies confirmed that the number of PMN adherent to hypoxic HUVEC or to the hypoxic umbilical vein endothelium was much greater than the number of PMN on normoxic endothelial cells. Both in vitro and ex vivo, PMN adherent to the hypoxic endothelial cells to the contrary of the ones adherent to normoxic endothelial cells demonstrated membrane foldings typical of an activated state. CONCLUSION: These results show that in a complete vein, hypoxia induced an increased adhesiveness of endothelial cells for PMN by a similar mechanism to the one observed for cultured endothelial cells. They suggest an active role of endothelial cells in the initiation of the inflammatory response often described in ischemic-reperfused organs.

Antibodies, Monoclonal↗

PubMed1994

Hypoxic human umbilical vein endothelial cells induce activation of adherent polymorphonuclear leukocytes.

Several pieces of evidence are reported for the accumulation of activated neutrophils in ischemic and reperfused tissues leading to the transformation of the ischemic tissue into an inflammatory territory and to an enhancement of tissue damages during reoxygenation. However, the molecular mechanisms responsible for these observations and the precise role played by endothelial cells in this process are still poorly understood. In this study, an in vitro model that mimics this situation was used to investigate the effects of hypoxia-incubated human umbilical vein endothelial cells (HUVEC) on polymorphonuclear leukocyte (PMN) functions. A strong PMN activation characterized by an increase in intracellular calcium concentration as well as by superoxide anion release and leukotriene B4 production was observed when these cells were coincubated with hypoxic HUVEC. On the other hand, conditioned medium from hypoxia-incubated HUVEC failed to activate PMN, as determined by the lack of PMN calcium concentration increase, the failure of superoxide anion production enhancement, as well as the absence of effects on the integrin CD18, CD11a, and CD11b expression. These results indicate that the presence of hypoxia-incubated HUVEC is necessary to obtain an activation of the PMN, probably via the adherence process. Once activated by coincubation with hypoxic HUVEC, PMN became cytotoxic, as evidenced by 51Cr released from prelabeled HUVEC. This cytotoxic effect of activated PMN for hypoxic endothelial cells could be prevented by a combination of superoxide dismutase and catalase (94% inhibition), whereas superoxide dismutase alone was inefficient. Antiprotease (alpha 2-macroglobulin) and a specific elastase inhibitor (MAAPV-CMK) were also inefficient. These results correlate very well with the fact that no increase in elastase release could be observed in supernatants from PMN coincubated with hypoxic HUVEC. Furthermore, when adherence process was blocked by oleic acid or by anti-ICAM-1 monoclonal antibodies, protection was, respectively, 90% and 72%. We thus evidenced that free radicals but not elastase released from activated PMN coincubated with hypoxic HUVEC are involved in HUVEC injury. We conclude from these results that PMN activation is initiated by PMN adherence to hypoxic HUVEC. These observations indicate that hypoxic HUVEC may be partly responsible for neutrophil activation observed in ischemic tissues, which is part of the amplification process of tissue damage.

Antigens, CD↗

PubMed1994

Hypoxia stimulates human endothelial cells to release smooth muscle cell mitogens: role of prostaglandins and bFGF.

Abnormal smooth muscle cell (SMC) proliferation is observed in several pathological situations such as atherosclerosis, pulmonary hypertension, and venous pathologies, resulting in a thickening of the vessel wall. If endothelial cells have been assumed to play a role in the triggering of this proliferation, no direct evidence is available. As ischemia is often linked to these situations, we exposed human umbilical vein endothelial cells (HUVEC) to hypoxia. The HUVEC-conditioned medium was then added to SMC and the proliferation of these cells was measured. We observed a pro-proliferative activity for SMC of the hypoxic HUVEC-conditioned medium but not of the normoxic HUVEC one. This pro-proliferative activity could not be inhibited if HUVEC were treated with cycloheximide but was blocked if the synthesis of prostaglandins by HUVEC was inhibited during hypoxia. PGD2, and especially PGF2 alpha at the concentration found in the hypoxic HUVEC-conditioned medium, were demonstrated to have a mitogenic effect on SMC. PGE2 also showed a pro-proliferative activity but at higher concentrations. In addition, the kinetics of increase in SMC proliferation induced by a mixture of the four prostaglandins at the corresponding concentrations was the same as the one observed with hypoxic HUVEC-conditioned medium. However, when tested on fibroblasts which do not respond to PGF2 alpha, hypoxic HUVEC-conditioned medium also had a pro-proliferative activity. In addition, anti-bFGF antibodies but not anti-PDGF blocked the mitogenic activity of this conditioned medium for SMC. Finally, the mitogenic effects of PGF2 alpha and of bFGF on SMC are additive. These results indicate that bFGF is probably also involved. These results indicate that these prostaglandins act in synergy with bFGF and are the molecules responsible for the pro-proliferative activity observed in hypoxic HUVEC-conditioned medium. We propose that these findings can explain the excessive growth of SMC in blood vessels following chronic ischemic situations.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1993

Stimulation of prostaglandin synthesis by human endothelial cells exposed to hypoxia.

In ischemic organs, arachidonic acid (AA) metabolites and mostly prostaglandins (PGs) have been found to be released in high amounts. The mechanism for this AA metabolism activation and its physiological implications are not clear. Because endothelial cells are an important source of PGs and because they seem to be very rapidly affected by ischemia, we developed an in vitro model where human endothelial cells were submitted to hypoxia. An important specific activation of phospholipase A2 was observed during hypoxia, which was concomitant with a rise in cytosolic calcium concentration. Endothelial cells synthetize in normal conditions as a mean 1.42, 1.00, 7.69, and 26.92 ng/mg proteins of, respectively, PGE2, PGD2, PGF2 alpha, PGI2. An important increase of about five- to ninefold in the synthesis of the four PGs was observed during hypoxia, which followed the same kinetics as the PLA2 activation. This increase in PG synthesis was sensitive to cyclooxygenase inhibitors. During reoxygenation, PG synthesis decreased back to the basal level of resting cells, suggesting that cells were able to recover their homeostasis after hypoxia. These observations indicate that endothelial cells exposed to oxygen deprivation are a major source of PGs and could contribute to the high amounts of PG released in vivo in ischemic organs.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗

PubMed1993

Increased PMN adherence on endothelial cells after hypoxia: involvement of PAF, CD18/CD11b, and ICAM-1.

Ischemia is a well-known situation occurring in several diseases. There is a large body of evidence for the accumulation of neutrophils in the microvascular injury and the transformation of ischemic tissue into an inflammatory territory. However, the molecular mechanisms underlying this phenomenon are still poorly understood. The effects of hypoxia were investigated on human umbilical vein endothelial cells (HUVEC) in culture, and a very strong activation of these cells was obtained with an induction of the platelet-activating factor (PAF) synthesis which was optimal after 90 min of hypoxia. PAF was chemically identified by gas chromatography-mass spectrometry. Along with incubation under hypoxia, a constant increase in the adherence of unstimulated human polymorphonuclear neutrophils (PMN) to endothelial cells was observed. The role of PAF and of adhesion glycoproteins in this hypoxia-induced neutrophil adherence to HUVEC was then assessed. The adherence was mediated by PAF after 90 min of hypoxia as indicated by the inhibition obtained with PAF receptor antagonists and with PAF synthesis inhibition. When tested on HUVEC incubated for 120 min under hypoxia, PAF antagonists could not inhibit the PMN adherence, whereas inhibition of PAF during hypoxia could block the process, suggesting a role of PAF acting as a second messenger. In addition, the inhibitory effects obtained using monoclonal antibodies indicate that this increased adherence was also mediated by intracellular adhesion molecule 1 on HUVEC and by CD18/CD11b on neutrophils. GMP-140 seems also to be involved after 90 min hypoxia but not after 120 min hypoxia, which correlated well with the presence of PAF.(ABSTRACT TRUNCATED AT 250 WORDS)

Antibodies, Monoclonal↗

PubMed1993

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.

Adenosine Triphosphate↗

PubMed1993

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.

Adenosine Triphosphate↗

PubMed1992

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.

3-Hydroxybutyric Acid↗

PubMed1992

Human umbilical vein endothelial cells submitted to hypoxia-reoxygenation in vitro: implication of free radicals, xanthine oxidase, and energy deficiency.

Ischemia-reperfusion is observed in various diseases such as myocardium infarct. Different theories have been proposed to explain the reperfusion injury, among them that the free radical generation plays a crucial role. To study the mechanisms of the reperfusion injury, a hypoxia (H)-reoxygenation (R) model upon human umbilical vein endothelial cells in culture was developed in order to mimic the in vivo situation. Different parameters were quantified and compared under H or H/R, and we found that oxygen readmission led to damage amplification after a short hypoxia period. To estimate the importance of various causes of toxicity, the effects of various protective molecules were compared. Different antioxidant molecules, iron-chelating agent, xanthine oxidase inhibitors, and energy-supplying molecules were very efficient protectors. Synergy could also be observed between the antioxidants and the energy-supplying molecules or the xanthine oxidase inhibitors. The toxic effect of O2.(-) could be lowered by the presence of SOD or glutathione peroxidase in the culture medium, whereas glutathione peroxidase was the most efficient enzyme when injected into the cells. The production of O2.(-) and of H2O2 by endothelial cells was directly estimated to be, respectively, of 0.17 and 0.035 mumol/min/mg prot during the R period. O2.(-) production was completely inhibited when allopurinol was added during H and R. In addition, a xanthine oxidase activity of 21.5 10(-6) U/mg prot could be observed by a direct assay in cells after H but not in control cells, thus confirming the previous conclusions of xanthine oxidase as a potent source of free radicals in these conditions. Thanks to the use of cultured human endothelial cells, a clear picture was obtained of the overall process leading to cell degenerescence during the reoxygenation process. We particularly could stress the importance of the low energetic state of these cells, which is a critical factor acting synergistically with the oxidant molecules to injure the cells. These results also open new possibilities for the development of new therapeutics for ischemia.

Cell Hypoxia↗

PubMedDate not supplied

[The relation between venous stasis and the occurrence of pain].

Venous stasis is a situation encountered commonly in varicose disorders. The potential implications of this decrease in oxygen levels in terms of the status of the cells of the vein were assessed. When endothelial cells are subjected to hypoxia, there is stimulation of the cells which shows itself as increased synthesis of prostaglandins and of PAF (Platelet Activating Factor). The synthesis of these typical mediators of inflammation results from activation by the calcium of phospholipase A2 which releases the arachidonic acid of phospholipids and this increase in intracellular calcium results itself from a fall in efficacy of calcium pumps due to the fall in ATP caused by hypoxia. Thus the fall in oxygen leads to the production of mediators of inflammation which activate leucocytes and result in local micro-inflammation which can be very rapidly eliminated if the circulation is restored but which can also cause irreversible damage to the vein by changes in venous tissue due to activated leucocytes which release proteases and free radicals after having penetrated the intima of the vein. These processes offer an explanation for the histological changes seen in varicose veins and the onset of localised pain during the development of such disease.

Humans↗

PubMedDate not supplied

[A comparative study of the protective effect of different phlebotonic agents on endothelial cells in hypoxia].

Numerous reported findings indicate that the etiology of venous diseases is multifactorial. One of the chief factors is certainly stasis of blood in the veins of the lower limbs during long periods spent standing up. This stasis causes tissue hypoxia which first affects the venous wall. Because of their location at the interface between blood and vein wall and because of their fragility, endothelial cells are the first to suffer from the lack of oxygen. With the aim of understanding these events, the authors have developed a model of endothelial cells in culture subjected to hypoxia in vitro which mimics the conditions encountered clinically. This model has enabled us to test various drugs commonly used in venous disease. These included GbE, the active ingredient of GINKOR FORT, diosmin and procyanidol oligomers. It was thus shown that only GbE was not toxic to endothelial cells. GbE was also found to be capable of effectively protecting cells exposed to hypoxia. Protection under the influence of diosmin was obtained only at concentrations very close to toxic doses. In contrast, procyanidol oligomers offered no protection. The protective effect of GbE is believed to be due to the action of terpenes on the energy metabolism of the cell.

Antihypertensive Agents↗

PubMedDate not supplied

[A comparative study of the protective effect of various phlebotonic agents on hypoxic endothelial cells].

Many findings indicate that the etiology of venous disease is multifactorial. One of the chief factors is certainly the stasis of blood in the lower limbs during long periods of standing. This stasis causes tissue hypoxia which first affects the venous wall. Because of their site at the blood/vein wall interface as well as their fragility, endothelial cells are the first to suffer from lack of oxygen. In order to understand these events, the authors have developed a model of endothelial cells in culture subjected to hypoxia in vitro which imitates conditions encountered clinically. This model was used to test different drugs commonly used in venous pathology. These included GbE, the active ingredient of GINKOR FORT, diosmine and procyanidolic oligomers. It was thus shown that only GbE was not toxic to endothelial cells. Furthermore, GbE was capable of effectively protecting cells subjected to hypoxia. Protection with diosmine was obtained only at concentrations very close to toxic doses. In contrast, procyanidolic oligomers afforded no protection. The protective effect of GbE is believed to be due to the action of terpenes on cellular energy metabolism.

Antihypertensive Agents↗