Biomedical subjects

C Michiels

Publications and source records attributed to C Michiels.

At least 19 recordsLinked to original sources

PubMed2002

Role of the endothelium and blood stasis in the appearance of varicose veins.

Today, chronic venous insufficiency affects millions of people but the investigation of veins and of venous diseases is still very poor. Additionally, the mechanism of the occurrence of varicose veins is not understood. Blood stasis is often associated with these pathological situations and we propose that resulting ischemic conditions can trigger the endothelium to release inflammatory mediators and growth factors. On one hand, the inflammatory mediators will recruit and activate neutrophils, which then infiltrate the venous wall and damage components of the extracellular matrix. On the other hand, growth factors induce smooth muscle cell migration, proliferation and de-differentiation into the synthetic phenotype, all together leading to the formation of neointima. These processes, being repeated over time, would eventually lead to alterations of the venous wall as observed in varicose veins. Venotropic drugs are used to treat chronic venous insufficiency. They are able to increase venous tone and to decrease vein and capillary permeability but they are also able to protect the endothelial cells against ischemia. Indeed, they target complexes of the mitochondrial respiratory chain and maintain ATP production during hypoxia. Hence, the cells are resistant to ischemia and do not release inflammatory mediators and growth factors. These drugs should thus be able to prevent the alterations of the venous wall induced by blood stasis.

Endothelium, Vascular↗

PubMed2001

Transduction pathways involved in Hypoxia-Inducible Factor-1 phosphorylation and activation.

Hypoxia-Inducible Factor-1 (HIF-1) is a transcription factor which is activated by hypoxia and involved in the adaptative response of the cell to oxygen deprivation. During hypoxic stress, HIF-1 triggers the overexpression of genes coding for glycolytic enzymes and angiogenic factors. To be active HIF-1 must be phosphorylated. HIF-1 is a substrate for various kinase pathways including PI-3K and the MAP kinases ERK and p38. Several transduction pathways have been proposed which act downstream of putative oxygen sensors and lead to the activation of these kinases. In this review, we summarize some of the latest advances describing the possible signaling pathways leading to HIF-1 phosphorylation and subsequent activation. The physiological relevance of these regulations is also discussed.

Angiogenesis Inhibitors↗

PubMed2001

Adenovirus-mediated gene transfer of human platelet-activating factor-acetylhydrolase prevents injury-induced neointima formation and reduces spontaneous atherosclerosis in apolipoprotein E-deficient mice.

BACKGROUND: Atherosclerosis is characterized by an early inflammatory response involving proinflammatory mediators such as platelet-activating factor (PAF)-like phospholipids, which are inactivated by PAF-acetylhydrolase (PAF-AH). The effect of adenovirus-mediated expression of PAF-AH on injury-induced neointima formation and spontaneous atherosclerosis was studied in apolipoprotein E-deficient mice. METHODS AND RESULTS: Intravenous administration of an adenovirus (5 x 10(8) plaque-forming units) directing liver-specific expression of human PAF-AH resulted in a 3.5-fold increase of plasma PAF-AH activity at day 7 (P<0.001); this was associated with a 2.4- and 2.3-fold decrease in malondialdehyde-modified LDL autoantibodies and the lysophosphatidylcholine/phosphatidylcholine ratio, respectively (P<0.001 for both). Non-HDL and HDL cholesterol levels in PAF-AH-treated mice were similar to those of control virus-treated mice. Seven days after virus injection, endothelial denudation of the common left carotid artery was induced with a guidewire. Neointima formation was assessed 18 days later. PAF-AH gene transfer reduced oxidized lipoproteins by 82% (P<0.001), macrophages by 69% (P=0.006), and smooth muscle cells by 84% (P=0.002) in the arterial wall. This resulted in a 77% reduction (P<0.001) of neointimal area. Six weeks after adenovirus-mediated gene transfer, spontaneous atherosclerotic lesions in the aortic root were analyzed. PAF-AH gene transfer reduced atherosclerotic lesions by 42% (P=0.02) in male mice, whereas a nonsignificant 14% reduction was observed in female mice. Basal and PAF-AH activity after gene transfer were higher in male mice than in female mice (P=0.01 and P=0.04, respectively). CONCLUSIONS: Gene transfer of PAF-AH inhibited injury-induced neointima formation and spontaneous atherosclerosis in apolipoprotein E-deficient mice. Our data indicate that PAF-AH, by reducing oxidized lipoprotein accumulation, is a potent protective enzyme against atherosclerosis.

1-Alkyl-2-acetylglycerophosphocholine Esterase↗

PubMed2001

c-JUN gene induction and AP-1 activity is regulated by a JNK-dependent pathway in hypoxic HepG2 cells.

Hypoxia is an important pathophysiological stress that occurs during blood vessel injuries and tumor growth. It is now well documented that hypoxia leads to the activation of several transcription factors which participate in the adaptive response of the cells to hypoxia. Among these transcription factors, AP-1 is rapidly activated by hypoxia and triggers bFGF, VEGF, and tyrosine hydroxylase gene expression. However, the mechanisms of AP-1 activation by hypoxia are not well understood. In this report, we studied the events leading to AP-1 activation in hypoxia. We found that c-jun protein accumulates in hypoxic HepG2 cells. This overexpression is concomitant with c-jun phosphorylation and JNK activation. Moreover, we showed that AP-1 is transcriptionally active. We also observed that AP-1 transcriptional activity is inhibited by a MEKK1 dominant negative mutant. Moreover, the MEKK1 dominant negative mutant as well as deletion of the AP-1 binding sites within the c-jun promoter inhibited the c-jun promoter activation by hypoxia. All together, these results indicate that, in hypoxic HepG2 cells, AP-1 is activated through a JNK-dependent pathway and that it is involved in the regulation of the c-jun promoter, inducing a positive feedback loop on AP-1 activation via c-jun overexpression.

Cell Hypoxia↗

PubMed2001

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+).

Adenosine Triphosphate↗

PubMed2001

Flocculation and coflocculation of bacteria by yeasts.

Biotransformations in natural environments frequently involve interactions between microorganisms. Although there are many reports on the interactions between bacteria, interactions between yeasts and bacteria have not been extensively studied. Previously we reported on the flocculation and coflocculation of Pediococcus damnosus by Saccharomyces cerevisiae. Now we report that several other yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, and Schizosaccharomyces pombe, induce flocculation with several industrially or medically relevant bacteria, including Bacillus subtilis, Pseudomonas aeruginosa, and Staphylococcus aureus. Candida utilis was one of the best flocculation inducers. The results are discussed with respect to interactions between yeasts and bacteria and their applications in industry and medicine.

Bacillus subtilis↗

PubMed2001

Heterologous expression of the Bacillus pumilus endo-beta-xylanase (xynA) gene in the yeast Saccharomyces cerevisiae.

The endo-beta-xylanase-encoding gene (xynA) of Bacillus pumilus PLS was isolated from a genomic DNA library and the open reading frame (ORF) was inserted in expression vectors for the yeast Saccharomyces cerevisiae. Plasmid pFN3 harboured the xynA ORF fused to the yeast mating pheromone alpha-factor signal sequence (MFalpha1s) under the control of the alcohol dehydrogenase II gene promotor (ADH2P) and terminator (ADH2T) sequences. In plasmid pFN4, the MFalpha1S-xynA ORF was brought under the control of the phosphoglycerate kinase I gene promotor (PGK1p) and terminator (PGK1T) sequences. Autoselective, recombinant S. cerevisiae [fur1::LEU2] strains bearing pFN3 or pFN4 secreted functional endo-beta-xylanase when grown in complex medium. Enzymatic activities in the culture supernatants reached maximum levels of 8.5 nkat/ml and 4.5 nkat/ml, respectively. The temperature and pH optimum for both the bacterial and the recombinant xylanase were 58 degrees C and pH 6.2.

Bacillus↗

PubMed2001

Coflocculation of Escherichia coli and Schizosaccharomyces pombe.

Several yeasts, such as Candida utilis, Dekkera bruxellensis, Hanseniaspora guilliermondii, Kloeckera apiculata, Saccharomyces cerevisiae and Schizosaccharomyces pombe, were found to coaggregate with Escherichia coli, but S. pombe showed much less coflocculation than the other yeasts (Peng et al. 2001)). S. pombe is known to have galactose-rich cell walls and we investigated whether this might be responsible for its different behavior by studying the wild-type TP4-1D, with a mannose to galactose ratio of 1 to 1.2, and the glycosylation mutant gms1delta (Man:Gal=1:0). The wild-type induced very low levels of coflocculation (3%) while gms1delta induced a remarkable amount of coflocculation (48%). Coflocculation of the mutant was inhibited by mannose but not affected by galactose or glucose. The S. cerevisiae mnn2 mutant, with a mannan structure similar to gms1delta, also showed a high degree of coflocculation (40%). However, S. cerevisiae mutant mnn9, with a mature core similar to S. pombe, showed decreased coflocculation (21.3%). Both these S. cerevisae mutants were sensitive to mannose inhibition. Coflocculation of E. coli and gms1delta also could be inhibited by gms1delta mannan and plant lectins, such as HHA, GNA and NPA, specific to either alpha-1-3- or alpha-1-6-linked mannosyl units. From these results we conclude that the E. coli lectins may have specificity for alpha-1-6- and alpha-1-3-linked mannose residues either in the outer chain or in the core of S. pombe, but in wild-type strains these mannose residues are shielded by galactose residues.

Carbohydrate Sequence↗

PubMed2001

PGF(2alpha), a prostanoid released by endothelial cells activated by hypoxia, is a chemoattractant candidate for neutrophil recruitment.

Despite increasing evidence supporting the involvement of neutrophils in ischemic and postischemic damages, the mechanisms underlying the early recruitment of these cells are not completely understood. In this report, the effects of conditioned media from hypoxic endothelial cells on neutrophil chemotaxis were investigated by biochemical and morphological studies. We showed that conditioned media collected from several endothelial cell origins submitted to hypoxia as well as ischemic rat liver perfusion liquids have a chemotactic activity for neutrophils. The role of various chemoattractant molecules like HETEs, platelet-activating factor, and cytokines such as interleukin-8 and interleukin-1 was examined in the same model. Chemotactic peptide contribution was ruled out as boiled conditioned media still trigger chemotaxis. However, cell treatment with cyclooxygenase inhibitors, neutralization of PGF(2alpha) biological activity with polyclonal antibodies, and the neutrophil preincubation with a specific PGF(2alpha) antagonist, all dramatically inhibited neutrophil chemotaxis. A strong chemoattractant effect of pure exogenous PGF(2alpha) or of a synthetic analog was also observed. The major effect of PGF(2alpha) on neutrophil chemotaxis was confirmed ex vivo in a rat liver perfusion ischemic model. These results suggest that PGF(2alpha), a prostanoid abundantly released by the endothelium of hypoxic or ischemic tissues, is a chemoattractant molecule that might be involved in the early recruitment of neutrophils in ischemic organs.

Animals↗

PubMed2001

Degradation of starchy endosperm cell walls in nongerminating sterilized barley by fungi.

Strains of fungi from different origins, including isolates of the natural microflora of barley, were screened for their ability to modify barley starchy endosperm cell walls in situ. In an initial step, fungi were selected that degrade the major component of the cell walls, that is, (1-->3),(1-->4)-beta-D-glucan, in vitro on artificial media. Nongerminating, sterilized barley, obtained by gamma-irradiation, was inoculated with such fungi and subjected to solid state fermentation under conditions resembling those of a traditional malting process. For some strains of fungi, a clear correlation between the production of endo-beta-glucanase and the friability of the treated kernels was found. Image analysis of Calcofluor stained longitudinal sections of barley kernels fermented with the endo-beta-glucanase producing strains showed that starchy endosperm cell walls were modified. As malt quality is inversely related to its (1-->3),(1-->4)-beta-D-glucan content, the selected strains have high potential to be used as starter cultures during malt production, contributing to the processing quality of the final product.

Aspergillus↗

PubMed2001

Improvement of malt modification by use of Rhizopus VII as starter culture.

The development of a selected starter culture on malting barley and its effects on malt quality aspects were studied. Application of Rhizopus sporangiospores in a malting process resulted in increased beta-glucanase and xylanase contents of the malting barley and improved starchy endosperm cell-wall degradation. Activation of the sporangiospores and optimization of the inoculation procedure led to a further increase in enzyme levels and to larger and more consistent impacts on cell-wall modification. Whereas the main effect of the starter culture on beta-glucan degradation was observed during malting, a further decrease in beta-glucan during mashing suggests that the microbial enzymes that survived the kilning step were active during mashing. Other quality aspects that were influenced by the starter culture activity were protein modification, wort color, and wort pH. The level of microbial enzymes produced was related to the amount of barley kernels infected with the starter culture.

Cell Wall↗

PubMed2001

HIF-1 and AP-1 cooperate to increase gene expression in hypoxia: role of MAP kinases.

HIF-1 is the main transcription factor responsible for increased gene expression in hypoxia: VEGF, erythropoietin, GLUT-1, and glycolytic enzymes are such target genes and all participate in the adaptative response of cells to hypoxia. AP-1 activation by hypoxia has also been demonstrated in several cell lines and it cooperates with HIF-1 for increasing VEGF gene transcription in hypoxia. Both HIF-1 and AP-1 activation by hypoxia seems to involve members of the MAP kinase family. Here, we summarize the data indicating that ERK and JNK are needed for activation of HIF-1 and AP-1, respectively.

Animals↗

PubMed2001

Decrease in cell surface galactose residues of Schizosaccharomyces pombe enhances its coflocculation with Pediococcus damnosus.

Pediococcus damnosus can coflocculate with Saccharomyces cerevisiae and cause beer acidification that may or may not be desired. Similar coflocculations occur with other yeasts except for Schizosaccharomyces pombe which has galactose-rich cell walls. We compared coflocculation rates of S. pombe wild-type species TP4-1D, having a mannose-to-galactose ratio (Man:Gal) of 5 to 6 in the cell wall, with its glycosylation mutants gms1-1 (Man:Gal = 5:1) and gms1Delta (Man:Gal = 1:0). These mutants coflocculated at a much higher level (30 to 45%) than that of the wild type (5%). Coflocculation of the mutants was inhibited by exogenous mannose but not by galactose. The S. cerevisiae mnn2 mutant, with a mannan content similar to that of gms1Delta, also showed high coflocculation (35%) and was sensitive to mannose inhibition. Coflocculation of P. damnosus and gms1Delta (or mnn2) also could be inhibited by gms1Delta mannan (with unbranched alpha-1,6-linked mannose residues), concanavalin A (mannose and glucose specific), or NPA lectin (specific for alpha-1,6-linked mannosyl units). Protease treatment of the bacterial cells completely abolished coflocculation. From these results we conclude that mannose residues on the cell surface of S. pombe serve as receptors for a P. damnosus lectin but that these receptors are shielded by galactose residues in wild-type strains. Such interactions are important in the production of Belgian acid types of beers in which mixed cultures are used to improve flavor.

Cell Wall↗

PubMed2000

TTRAP, a novel protein that associates with CD40, tumor necrosis factor (TNF) receptor-75 and TNF receptor-associated factors (TRAFs), and that inhibits nuclear factor-kappa B activation.

CD40 belongs to the tumor necrosis factor (TNF) receptor family. CD40 signaling involves the recruitment of TNF receptor-associated factors (TRAFs) to its cytoplasmic domain. We have identified a novel intracellular CD40-binding protein termed TRAF and TNF receptor-associated protein (TTRAP) that also interacts with TNF-R75 and CD30. The region of the CD40 cytoplasmic domain that is required for TTRAP association overlaps with the TRAF6 recognition motif. Association of TTRAP with CD40 increases profoundly in response to treatment of cells with CD40L. Interestingly, TTRAP also associates with TRAFs, with the highest affinity for TRAF6. In transfected cells, TTRAP inhibits in a dose-dependent manner the transcriptional activation of a nuclear factor-kappaB (NF-kappaB)-dependent reporter mediated by CD40, TNF-R75 or Phorbol 12-myristate 13-acetate (PMA) and to a lesser extent by TRAF2, TRAF6, TNF-alpha, or interleukin-1beta (IL-1beta). TTRAP does not affect stimulation of NF-kappaB induced by overexpression of the NF-kappaB-inducing kinase (NIK), the IkappaB kinase alpha (IKKalpha), or the NF-kappaB subunit P65/RelA, suggesting it acts upstream of the latter proteins. Our results indicate that we have isolated a novel regulatory factor that is involved in signal transduction by distinct members of the TNF receptor family.

Amino Acid Sequence↗

PubMed2000

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.

Cell Communication↗

PubMed2000

ERK activation upon hypoxia: involvement in HIF-1 activation.

Hypoxia-inducible factor-1 (HIF-1) is a transcription factor activated by hypoxia. The HIF-1 activation transduction pathway is poorly understood. In this report, we investigated the activation of extracellular regulated kinases (ERK) in hypoxia and their involvement in HIF-1 activation. We demonstrated that in human microvascular endothelial cells-1 (HMEC-1), ERK kinases are activated during hypoxia. Using dominant negative mutants, we showed that ERK1 is needed for hypoxia-induced HIF-1 transactivation activity. Moreover, using a kinase assay and Western blot experiments, we showed that HIF-1alpha is phosphorylated in hypoxia by an ERK-dependent pathway. These results evidence the role of mitogen-activated protein kinase in the transcriptional response to hypoxia.

Blotting, Western↗

PubMed2000

High dose rate brachytherapy for superficial cancer of the esophagus.

PURPOSE: We analyzed our experience with external radiotherapy, combined modality treatment, or HDR brachytherapy alone to limited esophageal cancers. METHODS AND MATERIALS: From 1991 to 1996, 25 patients with limited superficial esophagus carcinomas were treated by high dose rate brachytherapy. The mean age was 63 years (43-86 years). Five patients showed superficial local recurrence after external radiotherapy. Eleven patients without invasion of the basal membrane were staged as Tis. Fourteen patients with tumors involving the submucosa without spreading to the muscle were staged as T1. Treatment consisted of HDR brachytherapy alone in 13 patients, external radiotherapy and brachytherapy in 8 cases, and concomitant chemo- and radiotherapy in 4 cases. External beam radiation was administered to a total dose of 50 Gy using 2 Gy daily fractions in 5 weeks. In cases of HDR brachytherapy alone (13 patients), 6 applications were performed once a week. RESULTS: The mean follow-up is 31 months (range 24-96 months). Twelve patients received 2 applications and 13 patients received 6 applications. Twelve patients experienced a failure (48%), 11/12 located in the esophagus, all of them in the treated volume. One patient presented an isolated distant metastasis. In the patients treated for superficial recurrence, 4/5 were locally controlled (80%) by brachytherapy alone. After brachytherapy alone, 8/13 patients were controlled (61%). The mean disease-free survival is 14 months (1-36 months). Overall survival is 76% at 1 year, 37% at 2 years, and 14% at 3 years. Overall survival for Tis patients is 24% vs. 20% for T1 (p = 0.83). Overall survival for patients treated by HDR brachytherapy alone is 43%. One patient presented with a fistula with local failure after external radiotherapy and brachytherapy. Four stenosis were registered, two were diagnosed on barium swallowing without symptoms, and two required dilatations. CONCLUSION: High dose rate brachytherapy permits the treating of patients with superficial esophageal cancer with good tolerance. Early tumors, located in the mucosa, might be treated by HDR brachytherapy alone or by a combined modality treatment in which HDR brachytherapy can take place like a boost. This approach may cure localized recurrences.

Adenocarcinoma↗

PubMed2000

Effect of venotropic drugs on the respiratory activity of isolated mitochondria and in endothelial cells.

Several drugs used in the treatment of chronic peripheral ischaemic and venous diseases, i.e. aescine, Cyclo 3, Ginkor Fort, hydroxyethylrutosides, naftidrofuryl, naphthoquinone and procyanidolic oligomers, were tested on the mitochondrial respiratory activity. The results show that all these drugs protected human endothelial cells against the hypoxia-induced decrease in ATP content. In addition, they all induced a concentration-dependent increase in respiratory control ratio (RCR) of liver mitochondria pre-incubated with the drugs for 60 min. The drugs were divided into two groups according to their effects. The first group (A), comprising aescine, Ginkor Fort, naftidrofuryl and naphthoquinone, increased RCR by decreasing state 4 respiration rate. The second group of drugs (B), comprising hydroxyethylrutosides, procyanidolic oligomers and Cyclo 3, increased RCR by increasing state 3 respiration rate. The drugs of group A were able to prevent the inhibition of complexes I and III respectively by amytal and antimycin A while the first two drugs of group B increased adenine nucleotide translocase activity. Cyclo 3 inhibited the carbonylcyanide m-chlorophenyl hydrazone (mCCP)-induced uncoupling of mitochondrial respiration. None of these seven drugs could protect complexes IV and V, respectively, from inhibition by cyanide and oligomycin. When tested on endothelial cells the drugs of group A, in contrast to group B, prevented the decrease in ATP content induced by amytal or antimycin A. The present results suggest that the protective effects on mitochondrial respiration activity by these venotropic drugs may explain their protective effect on the cellular ATP content in ischaemic conditions and some of their beneficial therapeutic effect in chronic vascular diseases.

Adenosine Triphosphate↗