Ilse Smets

Texas Biomedical Research Institute, San Antonio, Texas, United States

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Publications (11)41.55 Total impact

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    ABSTRACT: Cholesterol synthesis and transport in oligodendrocytes are essential for optimal myelination and remyelination in pathological conditions such as multiple sclerosis. However, little is known about cholesterol homeostasis in the myelin-forming oligodendrocytes. Liver X receptors (LXRs) are nuclear oxysterol receptors that regulate genes involved in cholesterol homeostasis and may therefore play an important role in de- and remyelination. We investigated whether LXRs regulate cholesterol homeostasis in oligodendrocytes. mRNA expression of genes encoding LXR-α and LXR-β and their target genes (ABCA1, ABCG1, ABCG4, apoE, and LDLR) was detected in oligodendrocytes derived from both neonatal and adult rats using quantitative real-time PCR. The expression of LXR-β and several target genes was increased during oligodendrocyte differentiation. We further demonstrated that treatment of primary neonatal rat oligodendrocytes with the synthetic LXR agonist T0901317 induced the expression of several established LXR target genes, including ABCA1, ABCG1, apoE, and LDLR. Treatment of oligodendrocytes with T0901317 resulted in an enhanced cholesterol efflux in the presence of apolipoprotein A-I or high-density lipoprotein particles. These data show that LXRs are involved in regulating cholesterol homeostasis in oligodendrocytes.
    Journal of Neuroscience Research 01/2012; 90(1):60-71. DOI:10.1002/jnr.22743 · 2.73 Impact Factor
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    ABSTRACT: Statins have attracted interest as a treatment option for multiple sclerosis (MS) because of their pleiotropic antiinflammatory and immunomodulatory effects. However, contradictory results have been described when they are applied to oligodendrocytes (OLGs), the cell type predominantly affected in MS. In this study we focus on the in vitro effect of statins on process outgrowth in OLN-93 cells, a well-characterized OLG-derived cell line, and primary cultures of neonatal rat OLGs. Application of the lipophilic simvastatin, as low as 0.1-1 μM, disturbs process formation of both cell types, leading to less ramified cells. We show that both protein isoprenylation and cholesterol synthesis are required for the normal differentiation of OLGs. It is further demonstrated that the expression of 2',3'-cyclic-nucleotide-3' phosphodiesterase (CNP) and tubulin is lowered, concomitant with a reduction of membrane-bound CNP as well as tubulin. Therefore, we propose that lack of isoprenylation of CNP could help to explain the altered morphological and biochemical differentiation state of treated OLGs. Moreover, expression of specific myelin markers, such as myelin basic protein, myelin-associated glycoprotein, and myelin oligodendrocyte glycoprotein, was compromised after treatment. We conclude that simvastatin treatment has detrimental effects on OLG process outgrowth, the prior step in (re)myelination, thereby mortgaging long-term healing of MS lesions.
    Journal of Neuroscience Research 11/2010; 88(15):3361-75. DOI:10.1002/jnr.22490 · 2.73 Impact Factor
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    ABSTRACT: Detailed practical information is provided with emphasis on mapping cytosolic and mitochondrial pH, mitochondrial Na(+), and briefly also aspects related to mitochondrial Ca(2+) measurements in living cells, as grown on (un)coated glass coverslips. This chapter lists (laser scanning confocal) microscope instrumentation and setup requirements for proper imaging conditions, cell holders, and an easy-to-use incubator stage. For the daily routine of preparing buffer and calibration solutions, extensive annotated protocols are provided. In addition, detailed measurement and image analysis protocols are given to routinely obtain optimum results with confidence, while avoiding a number of typical pitfalls.
    Methods in molecular biology (Clifton, N.J.) 01/2010; 591:275-309. DOI:10.1007/978-1-60761-404-3_17 · 1.29 Impact Factor
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    ABSTRACT: Transfected Chinese hamster ovary cells were cultured on bare uncoated chemical vapor deposited thin nano- and microcrystalline diamond surfaces, hydrophobic hydrogen- and hydrophilic oxygen-terminated. Optical and biochemical analyses show that compared to glass controls, growth and viability were not significantly affected (one-way-analysis of variance, ANOVA). Based on two-way ANOVA analyses, neither grain size nor surface termination had a significant influence until 5 days post-seeding.
    Physica Status Solidi (A) Applications and Materials 08/2009; 206(9):2042 - 2047. DOI:10.1002/pssa.200982206 · 1.53 Impact Factor
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    ABSTRACT: OLN-93 cells, a cell line established from spontaneously transformed rat brain glial cultures, are used as a model for oligodendrocytes. These cells are known to undergo morphological changes upon serum deprivation. The objective of the present study is to investigate a possible correlation between these morphological changes and (1) the loss or gain of oligodendrocyte markers and (2) the electrophysiological properties of these cells. Using RT-PCR and immunocytochemistry, we demonstrate that the OLN-93 cell line expresses a broad range of markers (NG2, CNP, MAG, MOG) both when cultured in medium containing 10% or 0.5% fetal calf serum. Whole-cell patch-clamp recordings demonstrate that, regardless of the culture conditions, OLN-93 cells mainly express delayed-rectifying K+ currents, a characteristic of immature oligodendrocytes. These currents are in part mediated by the shaker family of voltage-gated potassium channels. Kv1.1 and Kv1.3-expression are present at the mRNA and at the protein levels, and functional evidence for Kv1.3 mediated currents was obtained by using the selective blocker margatoxin. Under low serum conditions, OLN-93 cells exhibit differentiation-like morphological changes. However, we provide evidence that these morphological modifications do not necessarily correlate with biochemical or functional changes. Based on these data, we conclude that the OLN-93 cell line can be situated at a developmental stage between a late pre-oligodendrocyte and a late immature oligodendrocyte, regardless of serum concentration.
    Journal of neuroscience methods 08/2009; 184(1):1-9. DOI:10.1016/j.jneumeth.2009.07.004 · 1.96 Impact Factor
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    ABSTRACT: Renal ischemia and subsequent reperfusion lead to changes in the regulation of hydrogen ions across the mitochondrial membrane. This study was designed to monitor pH changes in the cytosol and mitochondria of Madin-Darby Canine Kidney cells exposed to metabolic inhibition and subsequent recovery. A classical one-photon confocal imaging approach using the pH-sensitive fluorophore carboxy SNARF-1 was used to define specific loading, calibration, and correction procedures to obtain reliable cytosolic and mitochondrial pH values in living cells. Metabolic inhibition resulted in both cytosolic and mitochondrial acidification, with a more pronounced decrease of mitochondrial pH as compared to the cytosolic pH. Shortly after removing the metabolic inhibition, cytosolic pH did not recover, whereas mitochondrial pH slowly increased. Our method is applicable to other cell types provided that the mitochondria can be loaded with SNARF-1 and that the cells possess a mitochondria-free region to measure SNARF-1 in the cytosol.
    Kidney International 02/2008; 73(2):226-32. DOI:10.1038/ · 8.52 Impact Factor
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    ABSTRACT: In ischemic or hypoxic tissues, elevated cytosolic calcium levels can induce lethal processes. Mitochondria, besides the endoplasmic reticulum, play a key role in clearing excessive cytosolic Ca2+. In a previous study, it was suggested that the clearance of cytosolic Ca2+, after approximately 18 min of metabolic inhibition (MI) in renal epithelial cells, occurs via the reverse action of the mitochondrial Na+/Ca2+ exchanger (NCX). For further investigating the underlying mechanism, changes in the mitochondrial Na+ concentration ([Na+](m)) were monitored in metabolically inhibited MDCK cells. CoroNa Red, a sodium-sensitive fluorescence probe, was used to monitor [Na+]m. In the first 15 min of MI, a twofold increase of [Na+]m was observed reaching 113 +/- 7 mM, whereas the cytosolic Na+ concentration ([Na+]c) elevated threefold, to a level of 65 +/- 6 mM. In the next 45 min of MI, [Na+]m dropped to 91 +/- 7 mM, whereas [Na+]c further increased to 91 +/- 4 mM. The striking rise in [Na+]m is likely sufficient to sustain the driving force for mitochondrial Ca2+ uptake via the NCX. Furthermore, when CGP-37157, a specific inhibitor of the mitochondrial NCX, was applied during MI, the second-phase drop of [Na+]m was completely abolished. The obtained results support the hypothesis that the mitochondrial NCX reverses after approximately 15 min of MI. Moreover, because the cellular homeostasis can recover after MI, the mitochondria likely protect MDCK cells from injury during MI by the reversal of the mitochondrial NCX. This study is the first to report [Na+]m measurements in nonpermeabilized living cells.
    Journal of the American Society of Nephrology 10/2005; 16(12):3490-7. DOI:10.1681/ASN.2005010075 · 9.47 Impact Factor
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    ABSTRACT: In ischemic or hypoxic tissues, elevated Ca2+ levels have emerged as one of the main damaging agents among other Ca2+-independent mechanisms of cellular injury. Because mitochondria, besides the endoplasmic reticulum, play a key role in the maintainance of cellular Ca2+ homeostasis, alterations in the mitochondrial Ca2+ content ([Ca2+]m) were monitored in addition to changes in cytosolic Ca2+ concentration ([Ca2+]i) during metabolic inhibition (MI) in renal epithelial Madin-Darby canine kidney (MDCK) cells. [Ca2+]i and [Ca2+]m were monitored via, respectively, fura 2 and rhod 2 measurements. MI induced an increase in [Ca2+]i reaching 631+/-78 nM in approximately 20 min, followed by a decrease to 118+/-9 nM in the next approximately 25 min. A pronounced drop in cellular ATP levels and a rapid increase in intracellular Na+ concentrations in the first 20 min of MI excluded Ca2+ efflux in the second phase via plasma membrane ATPases or Na+/Ca2+ exchangers (NCE). Mitochondrial rhod 2 intensities increased to 434+/-46% of the control value during MI, indicating that mitochondria sequester Ca2+ during MI. The mitochondrial potential (deltapsim) was lost in 20 min of MI, excluding mitochondrial Ca2+ uptake via the deltapsim-dependent mitochondrial Ca2+ uniporter after 20 min of MI. Under Na+-free conditions, or when CGP-37157, a specific inhibitor of the mitochondrial NCE, was used, no drop in [Ca2+]i was seen during MI, whereas the MI-induced increase in mitochondrial rhod 2 fluorescence was strongly reduced. To our knowledge, this study is the first to report that in metabolically inhibited renal epithelial cells mitochondria take up Ca2+ via the NCE acting in the reverse mode.
    American journal of physiology. Renal physiology 05/2004; 286(4):F784-94. DOI:10.1152/ajprenal.00284.2003 · 3.30 Impact Factor
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    ABSTRACT: In renal ischemia, tubular obstruction induced by swelling of epithelial cells might be an important mechanism for reduction of the glomerular filtration rate. We investigated ischemic cell swelling by examining volume regulation of A6 cells during metabolic inhibition (MI) induced by cyanide and 2-deoxyglucose. Changes in cell volume were monitored by recording cell thickness (T(c)). Intracellular pH (pH(c)) measurements were performed with the pH-sensitive probe 5-chloromethyl-fluoresceine diacetate. T(c) measurements showed that MI increases cell volume. Cell swelling during MI is proportional to the rate of Na(+) transport and is not followed by a volume regulatory response. Furthermore, MI prevents the regulatory volume decrease (RVD) elicited by a hyposmotic shock. MI induces a pronounced intracellular acidification that is conserved during a subsequent hypotonic shock. A transient acidification induced by a NH(4)Cl prepulse causes a marked delay of the RVD in response to a hypotonic shock. On the other hand, acute lowering of external pH to 5, simultaneously with the hypotonic shock, allowed the onset of RVD. However, this RVD was completely arrested approximately 10 min after the initiation of the hyposmotic challenge. The inhibition of RVD appears to be related to the pronounced acidification that occurred within this time period. In contrast, when external pH was lowered 20 min before the hyposmotic shock, RVD was absent. These data suggest that internal acidification inhibits cellular volume regulation in A6 cells. Therefore, the intracellular acidification associated with MI might at least partly account for the failure of volume regulation in swollen epithelial cells.
    AJP Cell Physiology 09/2002; 283(2):C535-44. DOI:10.1152/ajpcell.00371.2001 · 3.67 Impact Factor
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    ABSTRACT: We report, for the epithelial Na+ channel (ENaC) in A6 cells, the modulation by cell pH (pHc) of the transepithelial Na+ current (INa), the current through the individual Na+ channel (i), the open Na+ channel density (No), and the kinetic parameters of the relationship between I(Na) and the apical Na+ concentration. The i and N) were evaluated from the Lorentzian INa noise induced by the apical Na+ channel blocker 6-chloro-3, 5-diaminopyrazine-2-carboxamide. pHc shifts were induced, under strict and volume-controlled experimental conditions, by apical/basolateral NH4Cl pulses or basolateral arrest of the Na+/H+ exchanger (Na+ removal; block by ethylisopropylamiloride) and were measured with the pH-sensitive probe 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein. The changes in pHc were positively correlated to changes in INa and the apically dominated transepithelial conductance. The sole pHc-sensitive parameter underlying INa was No. Only the saturation value of the INa kinetics was subject to changes in pHc. pHc-dependent changes in No may be caused by influencing Po, the ENaC open probability, or/and the total channel number, NT = No/Po.
    The American journal of physiology 10/1999; 277(3 Pt 1):C469-79. · 3.28 Impact Factor
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    ABSTRACT:  In high-resistance, salt-absorbing epithelia the apical amiloride-sensitive Na+ channel is the key site for regulation of salt and water balance. The saturation of macroscopic Na+ transport through these channels was investigated using A6 epithelial monolayers. The relation between transepithelial Na+ transport (I Na) and apical Na+ concentration ([Na+]ap) under short-circuit conditions was studied. Michaelis-Menten analysis of the saturable short-circuit current (I sc) yielded an apparent Michaelis-Menten constant (K m I ) of 5mmol/l and a maximal current (I max) of 8μA/cm2. The microscopic parameters underlying I Na, namely the single-channel current (i) and the open channel density (N o), were investigated by the analysis of current fluctuations induced by the electroneutral amiloride analogue CDPC (6-chloro-3,5-diaminopyrazine-2-carboxamide). A two-state model analysis yielded the absolute values of i (0.18±0.01pA) and N o (65.38±9.57 million channels/cm2 of epithelium) at [Na+]ap=110mmol/l containing 50μmol/l CDPC. Our data indicate that in A6 cells both i and N o depend on [Na+]ap. Between 3 and ≈20mmol/l the density of conducting pores, N o, decreases sharply and behaves again as an almost [Na+]ap-independent parameter at higher [Na+]ap. The single-channel current clearly saturates with an apparent Michaelis-Menten constant, K m i , of ≈17mmol/l. Thus, the [Na+]ap dependence of N o as well as the limited transport capacity of the amiloride-sensitive Na+ channel are both responsible for the saturation of I Na.
    Pflügers Archiv - European Journal of Physiology 02/1998; 435(5):604-609. DOI:10.1007/s004240050560 · 3.07 Impact Factor

Publication Stats

112 Citations
41.55 Total Impact Points


  • 2010
    • Texas Biomedical Research Institute
      San Antonio, Texas, United States
  • 2008
    • Hasselt University
      • Biomedical Research Institute (BIOMED)
      Hasselt, Flemish, Belgium
  • 2005
    • University of Szeged
      Algyő, Csongrád, Hungary
  • 2004
    • Transnationale Universiteit Limburg
      University Center, Virginia, United States