This article was downloaded by: [New York University] On: 25 April 2015, At: 17:44 Publisher: Routledge Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
Journal of the American College of Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uacn20
Oxysterol-induced endothelial cell dysfunction in culture. a
a
S Ramasamy , G A Boissonneault & B Hennig
a
a
Department of Nutrition and Food Science, University of Kentucky, Lexington 40506-0054. Published online: 02 Sep 2013.
To cite this article: S Ramasamy, G A Boissonneault & B Hennig (1992) Oxysterol-induced endothelial cell dysfunction in culture., Journal of the American College of Nutrition, 11:5, 532-538, DOI: 10.1080/07315724.1992.10718258 To link to this article: http://dx.doi.org/10.1080/07315724.1992.10718258
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Oxysterol-Induced Endothelial Cell Dysfunction in Culture Santhini Ramasamy, PhD, Gilbert A. Boissonneault, PhD, and Bernhard Hennig, PhD, FACN Departments of Nutrition and Food Science (S.R., B.H.) and Clinical Sciences (G.A.B.), University of Kentucky, Lexington
Downloaded by [New York University] at 17:44 25 April 2015
Key words: oxysterols, cholesterol, endothelial cells, barrier function, ATPases, enzymes Cholesterol oxidation products (oxysterols), such as cholestan-3/3,5a,6(3-triol (Triol), may be atherogenic by altering the barrier function of the vascular endothelium. We have shown that incubation of endothelial cell monolayers with Triol increased transendothelial albumin transfer (i.e., decreased barrier function) in a concentration- and time-dependent manner. Such dysfunction of endothelium could result from alterations in membrane characteristics, including changes in membrane-associated enzyme activities. To test this hypothesis, endothelial monolayers were treated with 20 μΜ Triol and the activities of selected membrane enzymes were measured at 0, 2, 4, 6, 12 and 24 hours. Calcium-adenosine triphosphatase (Ca++-ATPase) and sodium, potassium, magnesium-adenosine triphosphatase (Na+, K+, Mg++-ATPase) activities were significantly increased after 4 or 2 hours incubation with 20 μΜ Triol, respectively. 5'-nucleotidase activity was significantly elevated only after a 24-hour exposure to Triol, whereas there was no change in angiotensin-converting enzyme (ACE) activity in response to 20 μΜ Triol treatment at any time studied. Compared with all concentrations tested 40 μΜ Triol increased Ca++-ATPase activity most markedly, with a significant increase already after a 2-hour exposure. No major morphological changes were noted until 12 hours of exposure to 20 μΜ Triol; obvious cellular damage was observed by 24 hours. Cultures treated with Triol for 24 hours showed significant signs of toxicity, measured by an elevated [3H]adenine release, compared with control cultures. These data demonstrate that Triol alters the activity of certain membrane-bound enzymes, particularly Na+, K+, Mg++-ATPase and Ca++-ATPase. This alteration of membrane function may in part contribute to cellular dysfunction, decreasing the ability of the endothelium to act as a selectively permeable barrier to plasma components. Abbreviations: ACE = angiotensin-converting enzyme, CA++-ATPase = calciumadenosine triphosphatase, HMG-CoA = hydroxymethylglutaryl-coenzyme A, Na+, K+, Mg++-ATPase = sodium, potassium, magnesium-adenosine triphospha tase, Triol = cholestan-3/8,5a,60-triol
INTRODUCTION
Cholesterol feeding can lead to atherosclerosis in animal models. However, the exact mechanism of the relationship between cholesterol and atherosclerosis is not known. Loss of endothelial integrity, via injury to or dysfunction of the endothelium, has been implicated as one of the important early events leading to atherosclerosis [8,9]. Experimental evidence suggests that some oxysterols, but not pure cho lesterol, may be important effectors of atherosclerotic le sion formation [ 10,11 ]. Indeed, exposure of cultured endo thelial cell monolayers to Triol caused a time- and concen tration-dependent decrease in endothelial barrier function, expressed as an increase in the movement of albumin from the luminal to the albuminal side of endothelial monolayers [12]. Such an alteration of in vivo endothelial barrier
Cholesterol is present ubiquitously in mammalian tis sues and as such is considered essential for the formation and function of cellular membranes and for the production of bile acids and steroid hormones. Under certain condi tions, such as exposure to active oxygen species, cholesterol is oxidized and forms many biologically active oxidative derivatives known as oxysterols [1]. These oxysterols are found at low levels in cholesterol-rich processed foods [24], and are also known to be produced endogeneously [5,6] in biological systems. Among the oxysterols, cholestan3/8,5a,6/8-triol (Triol) is considered to be one of the most toxic in biological systems [7].
Addressreprintrequests to Bernhard Hennig, PhD, Cell Nutrition Group,Dept. of Nutrition and Food Science, 212 Funkhouser Building, University of Kentucky, Lexington, KY 40506-0054. Journal of the American College of Nutrition, Vol. 11, No., 5, 532-538 (1992) Published by the American College of Nutrition 532
Downloaded by [New York University] at 17:44 25 April 2015
Oxysterols and Endothelial Dysfunction function could contribute to atherogenesis by allowing an increased rate of penetration of low density lipoprotein and other plasma components into the sub-endothelial spaces [13]. The mechanism by which oxysterols alter endothelial barrier function is not clear. Alteration of membrane lipid composition can lead to alterations in the activities of membrane-related proteins such as enzymes, receptors and transport systems [14-16]. Thus far, however, there have been no reports on the effect of Triol on membrane-bound enzyme activities in vascular endothelial cell cultures. Peng and coworkers [17] reported that exposure of smooth muscle cells to Triol resulted in a depression of Na+, K+ATPase and 5'-nucleotidase activities. In contrast, Na+, K+-ATPase activity was found to be augmented in dog brain synaptosomes exposed in vitro to 19-hydroxycholesterol or 20a-hydroxycholesterol [18]. Therefore, the pres ent study examined endothelial barrier function and the specific activities of selected membrane-bound enzymes, i.e., Ca++-ATPase, Na+, K+, Mg++-ATPase, angiotensinconverting enzyme (ACE), and 5'-nucleotidase, at various time intervals after exposure to Triol. Also, the cytotoxicity of Triol to endothelial cultures was evaluated by cellular release of [3H]adenine release.
METHODS Endothelial cells were obtained from porcine pulmo nary artery and cultured in medium (M-199, Gibco Lab oratories, Grand Island, NY) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) as described [19]. The cells were determined to be endothelial by their characteristic cobblestone morphology and ACE activity. Cells from passages 10-25 were used for the experiments. Cell passage number had no effect on barrier function or the activities of the enzymes examined. Albumin Transfer Experiment Barrier function expressed as albumin transfer across endothelial monolayers was measured as described earlier [19]. Briefly, cells were seeded on polycarbonate filters (Nucleopore Corp, Pleasanton, CA), glued to polystyrene chemotactic chambers (ADAPS Ine, Dedham, MA), and placed into 24-well plates (Costar, Cambridge, MA). After 48 hours, when the cells reached confluency, monolayers were treated for up to 24 hours with M-199 plus 5% fetal bovine serum containing 0, 10, 20 or 40 μΜ Triol. Triol was dissolved in ethanol and this solution was added to M-199 containing 5% crystalline fatty acid-free bovine serum albumin (Sigma Chemical Co, St. Louis, MO). The final concentration of ethanol and bovine serum albumin never exceeded 0.1%. Control cultures received vehicle
without sterols. Albumin transfer across the endothelial monolayers was measured after exposure to various con centrations of Triol for periods up to 24 hours. Adenine Release Studies Cellular release of radiolabeled adenine was determined as described by Andreoli and coworkers [20]. Cells were labelled with 1 μ θ [3H]adenine/well (New England Nu clear, Boston, MA: 25 Ci/mmol) for 16-18 hours. Percent release was calculated as (DPMmedia/DPMceiis + DPMmedia) x 100. Enzyme Assays For analysis of enzyme activities endothelial cells were seeded in PI00 dishes (Corning Glassworks, Corning, NY). After a 48-hour incubation the cells were exposed to 0, 10, 20 or 40 μΜ Triol-containing media for 2, 4, 6, 12 or 24 hours. After incubation the cells were mechanically har vested using a cell scraper and the cells were sedimented and sonicated (Microson Ultrasonic Cell Disruptor, Farmingdale, NY) for 10 seconds at 4°C. The homogenate was first centrifuged at 4°C and 1500 rpm for 5 minutes, and the supernatant was used for the determination of enzyme activities. An aliquot from each homogenate was used for protein determination [21]. ACE, which is a marker enzyme for endothelial cells, was assayed according to the protocol supplied by Ventrex Laboratories (Portland, ME). The enzyme activity was calculated as units/mg protein. One unit was defined as the quantity of enzyme required to hydrolyze 1 % of the substrate per minute under incubation conditions. Na+, K+, Mg++-ATPase and Ca++-ATPase activities were measured according to the procedure described by Evans [22]. The inorganic phosphate liberated was meas ured as described by Fiske and Subbarow [23]. The enzyme activity was expressed as nmoles Pi liberated/mg protein/ hour. The method by Glastris and Pfeiffer [24] was used to assay for 5'-nucleotidase activity. The enzyme activity was expressed as nmoles of Pi liberated/mg protein/hour. Statistical Analysis Data were analyzed using SAS (Statistical Analysis Sys tem, Cary, NC). Comparisons between treatments were made by analysis of variance with post-hoc comparisons of the means made by Fischer's least significant difference method. Statistical probability of p < 0.05 was considered significant.
RESULTS Figure 1 shows the effect of a 24-hour incubation of endothelial cell monolayers with 0 (control), 10, 20, or 40
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION
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Oxysterols and Endothelial Dysfunction 30-1
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Journal of the American College of Nutrition Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/uacn20
Oxysterol-induced endothelial cell dysfunction in culture. a
a
S Ramasamy , G A Boissonneault & B Hennig
a
a
Department of Nutrition and Food Science, University of Kentucky, Lexington 40506-0054. Published online: 02 Sep 2013.
To cite this article: S Ramasamy, G A Boissonneault & B Hennig (1992) Oxysterol-induced endothelial cell dysfunction in culture., Journal of the American College of Nutrition, 11:5, 532-538, DOI: 10.1080/07315724.1992.10718258 To link to this article: http://dx.doi.org/10.1080/07315724.1992.10718258
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http:// www.tandfonline.com/page/terms-and-conditions
Oxysterol-Induced Endothelial Cell Dysfunction in Culture Santhini Ramasamy, PhD, Gilbert A. Boissonneault, PhD, and Bernhard Hennig, PhD, FACN Departments of Nutrition and Food Science (S.R., B.H.) and Clinical Sciences (G.A.B.), University of Kentucky, Lexington
Downloaded by [New York University] at 17:44 25 April 2015
Key words: oxysterols, cholesterol, endothelial cells, barrier function, ATPases, enzymes Cholesterol oxidation products (oxysterols), such as cholestan-3/3,5a,6(3-triol (Triol), may be atherogenic by altering the barrier function of the vascular endothelium. We have shown that incubation of endothelial cell monolayers with Triol increased transendothelial albumin transfer (i.e., decreased barrier function) in a concentration- and time-dependent manner. Such dysfunction of endothelium could result from alterations in membrane characteristics, including changes in membrane-associated enzyme activities. To test this hypothesis, endothelial monolayers were treated with 20 μΜ Triol and the activities of selected membrane enzymes were measured at 0, 2, 4, 6, 12 and 24 hours. Calcium-adenosine triphosphatase (Ca++-ATPase) and sodium, potassium, magnesium-adenosine triphosphatase (Na+, K+, Mg++-ATPase) activities were significantly increased after 4 or 2 hours incubation with 20 μΜ Triol, respectively. 5'-nucleotidase activity was significantly elevated only after a 24-hour exposure to Triol, whereas there was no change in angiotensin-converting enzyme (ACE) activity in response to 20 μΜ Triol treatment at any time studied. Compared with all concentrations tested 40 μΜ Triol increased Ca++-ATPase activity most markedly, with a significant increase already after a 2-hour exposure. No major morphological changes were noted until 12 hours of exposure to 20 μΜ Triol; obvious cellular damage was observed by 24 hours. Cultures treated with Triol for 24 hours showed significant signs of toxicity, measured by an elevated [3H]adenine release, compared with control cultures. These data demonstrate that Triol alters the activity of certain membrane-bound enzymes, particularly Na+, K+, Mg++-ATPase and Ca++-ATPase. This alteration of membrane function may in part contribute to cellular dysfunction, decreasing the ability of the endothelium to act as a selectively permeable barrier to plasma components. Abbreviations: ACE = angiotensin-converting enzyme, CA++-ATPase = calciumadenosine triphosphatase, HMG-CoA = hydroxymethylglutaryl-coenzyme A, Na+, K+, Mg++-ATPase = sodium, potassium, magnesium-adenosine triphospha tase, Triol = cholestan-3/8,5a,60-triol
INTRODUCTION
Cholesterol feeding can lead to atherosclerosis in animal models. However, the exact mechanism of the relationship between cholesterol and atherosclerosis is not known. Loss of endothelial integrity, via injury to or dysfunction of the endothelium, has been implicated as one of the important early events leading to atherosclerosis [8,9]. Experimental evidence suggests that some oxysterols, but not pure cho lesterol, may be important effectors of atherosclerotic le sion formation [ 10,11 ]. Indeed, exposure of cultured endo thelial cell monolayers to Triol caused a time- and concen tration-dependent decrease in endothelial barrier function, expressed as an increase in the movement of albumin from the luminal to the albuminal side of endothelial monolayers [12]. Such an alteration of in vivo endothelial barrier
Cholesterol is present ubiquitously in mammalian tis sues and as such is considered essential for the formation and function of cellular membranes and for the production of bile acids and steroid hormones. Under certain condi tions, such as exposure to active oxygen species, cholesterol is oxidized and forms many biologically active oxidative derivatives known as oxysterols [1]. These oxysterols are found at low levels in cholesterol-rich processed foods [24], and are also known to be produced endogeneously [5,6] in biological systems. Among the oxysterols, cholestan3/8,5a,6/8-triol (Triol) is considered to be one of the most toxic in biological systems [7].
Addressreprintrequests to Bernhard Hennig, PhD, Cell Nutrition Group,Dept. of Nutrition and Food Science, 212 Funkhouser Building, University of Kentucky, Lexington, KY 40506-0054. Journal of the American College of Nutrition, Vol. 11, No., 5, 532-538 (1992) Published by the American College of Nutrition 532
Downloaded by [New York University] at 17:44 25 April 2015
Oxysterols and Endothelial Dysfunction function could contribute to atherogenesis by allowing an increased rate of penetration of low density lipoprotein and other plasma components into the sub-endothelial spaces [13]. The mechanism by which oxysterols alter endothelial barrier function is not clear. Alteration of membrane lipid composition can lead to alterations in the activities of membrane-related proteins such as enzymes, receptors and transport systems [14-16]. Thus far, however, there have been no reports on the effect of Triol on membrane-bound enzyme activities in vascular endothelial cell cultures. Peng and coworkers [17] reported that exposure of smooth muscle cells to Triol resulted in a depression of Na+, K+ATPase and 5'-nucleotidase activities. In contrast, Na+, K+-ATPase activity was found to be augmented in dog brain synaptosomes exposed in vitro to 19-hydroxycholesterol or 20a-hydroxycholesterol [18]. Therefore, the pres ent study examined endothelial barrier function and the specific activities of selected membrane-bound enzymes, i.e., Ca++-ATPase, Na+, K+, Mg++-ATPase, angiotensinconverting enzyme (ACE), and 5'-nucleotidase, at various time intervals after exposure to Triol. Also, the cytotoxicity of Triol to endothelial cultures was evaluated by cellular release of [3H]adenine release.
METHODS Endothelial cells were obtained from porcine pulmo nary artery and cultured in medium (M-199, Gibco Lab oratories, Grand Island, NY) containing 10% fetal bovine serum (Hyclone Laboratories, Logan, UT) as described [19]. The cells were determined to be endothelial by their characteristic cobblestone morphology and ACE activity. Cells from passages 10-25 were used for the experiments. Cell passage number had no effect on barrier function or the activities of the enzymes examined. Albumin Transfer Experiment Barrier function expressed as albumin transfer across endothelial monolayers was measured as described earlier [19]. Briefly, cells were seeded on polycarbonate filters (Nucleopore Corp, Pleasanton, CA), glued to polystyrene chemotactic chambers (ADAPS Ine, Dedham, MA), and placed into 24-well plates (Costar, Cambridge, MA). After 48 hours, when the cells reached confluency, monolayers were treated for up to 24 hours with M-199 plus 5% fetal bovine serum containing 0, 10, 20 or 40 μΜ Triol. Triol was dissolved in ethanol and this solution was added to M-199 containing 5% crystalline fatty acid-free bovine serum albumin (Sigma Chemical Co, St. Louis, MO). The final concentration of ethanol and bovine serum albumin never exceeded 0.1%. Control cultures received vehicle
without sterols. Albumin transfer across the endothelial monolayers was measured after exposure to various con centrations of Triol for periods up to 24 hours. Adenine Release Studies Cellular release of radiolabeled adenine was determined as described by Andreoli and coworkers [20]. Cells were labelled with 1 μ θ [3H]adenine/well (New England Nu clear, Boston, MA: 25 Ci/mmol) for 16-18 hours. Percent release was calculated as (DPMmedia/DPMceiis + DPMmedia) x 100. Enzyme Assays For analysis of enzyme activities endothelial cells were seeded in PI00 dishes (Corning Glassworks, Corning, NY). After a 48-hour incubation the cells were exposed to 0, 10, 20 or 40 μΜ Triol-containing media for 2, 4, 6, 12 or 24 hours. After incubation the cells were mechanically har vested using a cell scraper and the cells were sedimented and sonicated (Microson Ultrasonic Cell Disruptor, Farmingdale, NY) for 10 seconds at 4°C. The homogenate was first centrifuged at 4°C and 1500 rpm for 5 minutes, and the supernatant was used for the determination of enzyme activities. An aliquot from each homogenate was used for protein determination [21]. ACE, which is a marker enzyme for endothelial cells, was assayed according to the protocol supplied by Ventrex Laboratories (Portland, ME). The enzyme activity was calculated as units/mg protein. One unit was defined as the quantity of enzyme required to hydrolyze 1 % of the substrate per minute under incubation conditions. Na+, K+, Mg++-ATPase and Ca++-ATPase activities were measured according to the procedure described by Evans [22]. The inorganic phosphate liberated was meas ured as described by Fiske and Subbarow [23]. The enzyme activity was expressed as nmoles Pi liberated/mg protein/ hour. The method by Glastris and Pfeiffer [24] was used to assay for 5'-nucleotidase activity. The enzyme activity was expressed as nmoles of Pi liberated/mg protein/hour. Statistical Analysis Data were analyzed using SAS (Statistical Analysis Sys tem, Cary, NC). Comparisons between treatments were made by analysis of variance with post-hoc comparisons of the means made by Fischer's least significant difference method. Statistical probability of p < 0.05 was considered significant.
RESULTS Figure 1 shows the effect of a 24-hour incubation of endothelial cell monolayers with 0 (control), 10, 20, or 40
JOURNAL OF THE AMERICAN COLLEGE OF NUTRITION
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Oxysterols and Endothelial Dysfunction 30-1
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