Lipids

Effect of Vitamin E on Linoleic Acid-MediatedInduction of Peroxisomal Enzymes in Cultured PorcineEndotheiial Cells1

BERNHARD HENNIG,* f2 GILBERT A. BOISSONNEAULT,**f CHIHG K. CHOW,*f YINWANG, t DANIEL H. MATULIONISf ANDHOWARD P. GLAUERT*f

Departments of *Nutrition and Food Science, **Clinical Sciences and fAnatomy and Neurobiology and

the ^Graduate Center for Toxicology, University of Kentucky, Lexington, KY 40506

ABSTRACT Linoleic acid decreases endothelial barrier function in culture. We hypothesize that the mechanism may involve induction of peroxisomes, withsubsequent generation of hydrogen peroxide, and thatvitamin E may protect against barrier function loss bypreventing the induction of peroxisomal enzymes. Toinvestigate this hypothesis, we exposed culturedendothelial cells to 0 or 90 umol/L linoleic acid[18:2(n-6)j, with or without 25 umol/L supplementalvitamin E, for 5 d. The induction of peroxisomes bylinoleic acid exposure was determined by measuringcellular peroxisomal ß-oxidationand catalase activity.Vitamin E alone had no effect on ß-oxidationor catalase activity, whereas linoleic acid exposure significantly increased both compared with control values.Vitamin E supplementation prevented induction of peroxisomal ß-oxidationand catalase activity by 18:2. Incontrast, cell enrichment with vitamin E had no effecton 18:2-induced accumulation of cytoplasmic lipid-Iikedroplets. These results confirm our hypothesis that theprotective effects of vitamin E against fatty acid-mediated endothelial cell injury may be due in part to theability of vitamin E to prevent the induction of peroxisomal ß-oxidationenzymes and thus the formation ofexcess hydrogen peroxide. J. /Vuir. 120:331-337,1990.

INDEXING KEY WORDS:

•endothelial cells •vitamin E •fatty acids•peroxisomes •cell injury

The mechanism of atherosclerosis may involve damage to or dysfunction of the vascular endothelium, withsubsequent reduced effectiveness of the endothelium asa barrier to plasma components (1, 2). High levels oftriglyceride-rich lipoproteins have been implicated inthe endothelial injury process (3, 4). Endothelial exposure to excessive amounts of free fatty acid anions,derived from the hydrolysis of triglyceride-rich lipopro

teins, may result in the accumulation of these fattyacids within endothelial cells and may lead to cellularchanges resulting in cell injury or dysfunction. This mayin turn allow increased penetration of remnant lipoproteins into the arterial wall (3-5). In support of thispossibility, we showed that exposure to oleic [18:l(n-9)](6)and especially to linoleic [18:2(n-6)],but not to linole-nic [18:3(n-3(](7),acid increased the transfer of albuminacross cultured porcine endothelial cell monolayers.Prior cellular incubation with media containing 25u.mol/L vitamin E for 24 h protected endothelial cellsfrom injury by pure linoleic acid and linoleic acidhydroperoxide, as indicated by a decreased rate of albumin transfer across the endothelium (8). The mechanism of the linoleic acid-specific increase in albumintransfer is not clear.

One possible mechanism of linoleic acid-inducedendothelial cell toxicity may involve peroxisomes,cytoplasmic organdíespresent in most if not all cells(9). Several types of chemicals, such as hypolipidemicdrugs, and nutritional alterations, such as feeding high-fat diets, have been shown to induce hepatic peroxisomal proliferation in rodents and other species (10-13).The peroxisomal fatty acid ß-oxidationpathway wasspecifically induced by peroxisome proliferators,whereas other enzymes, such as catalase, were slightlyinduced (10-13). Because hydrogen peroxide is an end-product of peroxisomal ß-oxidation,it has been hypothesized that the adverse effects of peroxisome

'Supported in part by grants 1PO1 HL36552 and 1RO1 CA43719

from the National Institutes of Health, a grant from the AmericanHeart Association, Kentucky Affiliate, and the Kentucky AgriculturalExperiment Station.

2To whom correspondence and reprint requests should be addressed.

0022-3166/90 $3.00 ©1990 American Institute of Nutrition. Received 24 July 1989.Accepted 27 November 1989.

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332 HENNIG ET AL.

proliferators, such as hepatic tumors, are caused by theoverproduction of hydrogen peroxide (10). We recentlydemonstrated that peroxisomal ß-oxidationand catalaseactivity are induced in cultured endothelial cells by thehypolipidemic drug ciprofibrate and by certain fattyacids (14, 15). Compared to enrichment with stearic(18:0),oleic and linolenic acid, cellular enrichment withlinoleic acid induced peroxisomal ß-oxidationmostmarkedly (15). It therefore seems possible that a metabolic relationship may exist between 18:2-induced peroxisomal ß-oxidationand the observed 18:2-inducedincrease in the transfer of albumin across cultured cellmonolayers.

Vitamin E deficiency results in peroxisome prolifera-tion in the liver (16). More specifically, vitamin E deficiency in rats induced the activities of peroxisomal fattyacid CoA oxidase and carnitine acyltransferases, but notof catalase (16). Furthermore, supplementation withvitamin E decreases the production of hydrogen peroxide from human polymorphonuclear leukocytes (17,18 ).Because vitamin E appears to be able to protect endothelial cells against 18:2-induced injury (8), the presentstudy was designed to investigate whether this protective action of vitamin E may be due in part to its abilityto prevent the induction of peroxisomal enzymes byfatty acids and thus to curtail the formation of cytotoxichydrogen peroxide. Endothelial cells were exposed tolinoleic acid in the presence or absence of vitamin E for5 d. We then quantified the activities of the peroxisomalenzyme catalase and the peroxisomal fatty acid ß-oxidation pathway. Ultrastructural changes also were assessed.

MATERIALS AND METHODS

Cell culture. Endothelial cells from porcine pulmonary arteries were obtained as described by Hennig et al.(6). Briefly, blood vessels were resected under sterileconditions and rinsed with medium 199 (M-199,GIBCOLaboratories, Grand Island, NY) containing antibiotics;the lumen was then exposed to 0.1% collagenase(Worthington Biochemical, Freeland, NJ)for 5 to 10min.After several rinses with M-199, the lumen was swabbedgently with a sterile, cotton-tipped applicator. The adherent endothelial cells were released into M-199 containing 10% fetal bovine serum (HyClone Laboratories,Logan, UT) and seeded into T-25 flasks. The flasks werecapped loosely and placed in a CO2 incubator (5%) at37°C.The medium was changed after 24 h, and cultures

were left in the incubator for approximately 5 to 8 d,when confluency was reached. The cells were thensubcultured in M-199 containing 10% fetal bovineserum by using standard techniques and flasks obtainedfrom Costar (Cambridge, MA). Cultures were determined to be endothelial by uniform morphology and byquantitative determination of angiotensin-converting

enzyme activity. Cells from passages 10-15 were usedin this study. Experiments were performed three times,and results were similar at cell passages 10-15. For thedetermination of fatty acid ß-oxidationand catalaseactivity, cells were plated on T-75 flasks or P-100 dishes(Costar). For electron microscopy studies, cells wereplated on gelatin-impregnated polycarbonate filters(Nucleopore, Pleasanton, CA; 13 mm diameter and 0.8jim pore size) glued to polystyrene chemotactic chambers (ADAPS, Dedham, MA) (6).

The experimental media were composed of M-199enriched with 5% fetal bovine serum, 90 umol/L (2.5mg/100 mL)high-purity (>99%) linoleic acid (Nu-Chek-Prep, Elysian, MN) and either 0 umol/L or 25 umol/L(1.1 mg/100 mL) vitamin E (ot-tocopherol). Fatty acid-and vitamin E-enriched culture media were prepared asdescribed earlier (6, 8).

Experimental procedures. Cells seeded for 4 h inmedium without supplemental vitamin E or fatty acidswere exposed to experimental media for 5 d to allow formaximal induction of peroxisomes. The experimentalmedia were composed of M-199 enriched with 5% fetalbovine serum (control media) plus either 25 umol/Lvitamin E, 90 umol/L 18:2, or 25 umol/L vitamin E and90 umol/L 18:2. Cells were harvested by first washingthem in ice-cold phosphate-buffered saline (PBS)andthen scraping with silicone rubber blades. Harvestedcells were centrifuged, resuspended in PBS and sonicated, all in ice-cold buffer. Homogenates were thenassayed for protein (19),catalase activity (20)or peroxisomal fatty acid ß-oxidation(21).To monitor vitamin Elevels in the media and cells as they may relate tobiological function, cell homogenates and media wereassayed for vitamin E (22, 23) after incubation for 0, 2and 5 d. For vitamin E determination, the method ofCatignani (22) was modified by redissolving the lipidresidue in methanol, by using 100% methanol as themobile phase of the high performance liquid chromatog-raphy system and by implementing the fluorescencedetection method of Hatam and Kayden (23).

Ultrastructural assessment involved the examination of 1}cells plated in control medium on gelatin-impregnated polycarbonate filters; 2) cells plated on filtersin control medium supplemented with 18:2(90umol/L);and 3) cells cultured in control medium supplementedwith vitamin E (25 umol/L) and 18:2 (90 umol/L). Cellsgrown in control medium supplemented with vitaminE alone also were examined. Exposure to 18:2, vitaminE, and vitamin E plus 18:2 was for 5 d. The cells on thefilters were fixed in 3.5% cacodylate buffered glutaral-dehyde fixative (pH 7.2-7.4) for 10 min at room temperature and then for an additional 80 min at 4°C.The

remaining tissue processing procedures were similar tothose detailed elsewhere (24).All epoxy-embedded tissues were sectioned at 80 to 100nm. From 30 to 50 cellsfrom four to nine filters per treatment were imaged orexamined in a Philips 400 electron microscope at 60 kV.

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FIGURE1 Effect of linoleic acid and vitamin Eon peroxi-somal ß-oxidationin cultured endothelial cells. Peroxisomalß-oxidationis expressed as nmol palmityl CoA oxidized permin per mg protein. The experimental media consisted ofM-199 enriched with 5% fetal bovine serum (Control) pluseither 25 umol/L vitamin E (VitE), 90 umol/L linoleic acid(18:2) or 25 umol/L vitamin E and 90 umol/L linoleic acid.Values are means ±SEM(n=6}.Data were analyzed by ANOVA,and when a significant difference occurred, a protected LSDtest was performed. Value for linoleic acid was significantlygreater than that for controls (p < 0.05).

Statistical analysis. Data were analyzed statisticallyby analysis of variance, and when significant differencesoccurred, a protected LSD test was performed (25). Astatistical probability of p < 0.05 was considered significant.

RESULTS

The effects of linoleic acid and vitamin E on peroxi-somal ß-oxidationin cultured endothelial cells areshown in Figure 1. The induction of peroxisomal ß-oxidation was not affected by vitamin E alone. Exposure to18:2, however, significantly increased ß-oxidationcompared with that in control cultures. This increase inperoxisomal ß-oxidationwas prevented by simultaneous enrichment with vitamin E.The chosen fatty acidconcentration did not appear to adversely affect cellviability as established by trypan blue exclusion studiesand morphological assessment by phase contrast microscopy, which showed maintenance of a typical cobblestone appearance.

The effects of 18:2 and vitamin E on peroxisomalcatalase activity are shown in Figure 2. As with theß-oxidationdata, 18:2 increased catalase activity compared with that in control cultures. Furthermore, simultaneous enrichment and vitamin E also prevented the

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FIGURE2 Effectof linoleic acid and vitamin Eon peroxisomal catalase activity in cultured endothelial cells. Catalaseactivity is expressed as umol hydrogen peroxide decomposedper min per mg protein. The experimental media consisted ofM-199 enriched with 5% fetal bovine serum (Control) pluseither 25 umol/L vitamin E (VitE), 90 umol/L linoleic acid(18:2) or 25 umol/L vitamin E and 90 umol/L linoleic acid.Values are means ±SEM(n=6}.Data were analyzed by ANOVA,and when a significant difference occurred, a protected LSDtest was performed. Value for linoleic acid was significantlygreater than that for controls (p < 0.05).

fatty acid-induced increase in catalase activity.Figure3 shows the levels of vitamin E in media and

cells. Cell enrichment with vitamin E appeared to increase throughout the 5-d incubation period. As cellsbecame enriched, vitamin E levels in the media fell. Incontrast to media enrichment with vitamin E alone,enrichment with both vitamin E and 18:2 led to acomplete depletion of vitamin E from the media after 5d. Conversely, cells exposed to both 18:2 and vitamin Eremained significantly enriched with vitamin E compared with control cultures, even after 5 d. The vitaminE level of endothelial cells immediately after plating,that is, before exposure to experimental media, was0.037 ±0.001 ug/mg protein.

The ultrastructure of cells grown on polycarbonatefilters in control culture medium suggested viable, synthetically active and well-preserved cells (Fig.4A). Thecells formed a monolayer, although occasional partialoverlapping was observed. Infrequently, small amountsof cellular debris were present between the cells and thepolycarbonate filters. The cell surface was uniform (Fig.4A}. Vesicular invaginations were uncommon. Themost prominent cytoplasmic components were heterogeneous lysosome-like bodies, rough endoplasmic retic-ulum, mitochondria, polyribosomes and, when in theplane of section, Golgi complex (Fig. 4A). Lipid-likedroplets were noted in only very few of the control

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FIGURE 3 Measurements of vitamin E levels in media (toppanel) and cells (bottom panel) after 2 or 5 d. The experimentalmedia consisted of M-199 enriched with 5% fetal bovineserum (Control) plus either 25 umol/L vitamin E (VitE), 90umol/L linoleic acid (18:2) or 25 umol/L vitamin E and 90umol/L linoleic acid. Values are means ±SEM(n = 6).

cultured cells. Nuclei were elongated and formedmostly of euchromatin, and they contained prominentnucleoli. The ultrastructure of cells grown in controlmedium supplemented with vitamin E was similar to

that of cells grown in control medium alone. However,the vitamin E-supplemented cultures contained morecellular debris than unsupplemented cultures. The morphology of cells cultured in medium to which 18:2 (90umol/L) was added deviated from that of cells grown incontrol medium in two main respects. These cells appeared larger and contained markedly more cytoplasmiclipid-like droplets than those grown in control medium(Fig.4B).With one exception, the structure of cell mono-layers grown in medium supplemented with vitamin Eand 18:2 was similar to that of cells treated with 18:2alone: Monolayers exposed to vitamin E and 18:2 contained considerably more intercellular material than18:2-treated cultures (Fig.4C). The debris often elevatedthe viable cells from the polycarbonate filters (Fig.4C).

DISCUSSION

Damage to or dysfunction of the vascular endothe-lium and the resulting disturbance in endothelial integrity may be involved in the process of atheroscleroticlesion formation (1, 2). High levels of triglyceride-richlipoproteins and the free fatty acids derived from triglycéridehydrolysis have been implicated in the injuryprocess of the endothelium and in a decrease in endothelial barrier function (3,4). In support of this possibility, we have shown that exposure to oleic (6) andespecially to linoleic, but not to linolenic (7), acid increased the transendothelial movement of albuminacross cultured cell monolayers.

The mechanism of the linoleic acid-specific increasein albumin transfer is not known at present. In additionto a selective accumulation of this fatty acid in cellularphospholipid and triglycéridefractions (7),we speculatethat metabolites of peroxisomal fatty acid ß-oxidation,such as hydrogen peroxide, may be involved in themechanism of endothelial cell injury (15)and the resulting decrease in the endothelial barrier function that wasobserved. There is evidence that high-fat diets induceperoxisomal ß-oxidationin the liver (12, 13) and thatpolyunsaturated fatty acids appear to be used preferentially for total cell oxidation (26). In the present study,exposing cultured endothelial cells to linoleic acidmarkedly increased peroxisomal ß-oxidation.Other 18-carbon fatty acids, such as stearic, oleic and linolenicacid, did not exhibit such a consistently marked influence on ß-oxidation(15).Whether the twofold increasein catalase activity is sufficient to detoxify the hydrogenperoxide produced by peroxisomal ß-oxidationis unclear.

FIGURE 4 Transmission electron micrographs of endothelial cell monolayers cultured on polycarbonate micropore filters.Cells were grown in control medium (A), or they were supplemented with 90 umol/L 18:2 (linoleic acid) (B) or with 25 umol/Lvitamin E and 90 umol/L 18:2 (C). Note that fatty acid-treated cells contain considerably more lipid-like droplets (Lp) (lipiddissolved during processing). Cells treated with both vitamin E and fatty acid were characterized by the presence of intercellulardebris (D). Lysosome-like structures, L; Golgi complex, G; mitochondria, M; nucleus, N; nucleolus, Nu; polycarbonate filter, F;rough endoplasmic reticulum, arrow; polyribosomes, arrowhead. Calibration bar in the lower left corner is equal to 1 urn (x 16,800).

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336 HENNIG ET AL.

Vitamin E may be antiatherogenic by protecting en-dothelial cells from fatty acid- and from fatty acidhydroperoxide-induced cell injury (8). Although the

mode of cell protection by vitamin E is not clear, itsbeneficial action might be via its antioxidant activity(27), its ability to act as a biological membrane stabilizer(28, 29), or its regulatory function in cell turnover rate(30). Furthermore, supplementation with vitamin E hasbeen shown to decrease the production of hydrogenperoxide from human polymorphonuclear leukocytes(17,18). Previous work from our laboratory showed thatsupplemental vitamin E protected cultured endothelialcell monolayers from linoleic acid-induced loss of bar

rier function (8).In this study we demonstrated that cells remained

enriched with vitamin E as long as 5 d following a singlevitamin E supplementation, allowing vitamin E to beavailable for its biological function as a protector againstfatty acid-induced cell injury. Results from the present

study suggest that one possible mechanism of vitaminE's action is to reduce the induction of peroxisomal

ß-oxidationenzymes and thus the formation of excesshydrogen peroxide. Vitamin E could reduce 18:2-in-duced peroxisomal ß-oxidation enzyme production bypreventing excess fatty acid from entering the cell andpreventing cellular accumulation of lipid-like droplets.

However, our data do not support this mechanism because ultrastructural evaluation of vitamin E-enriched

cells, which also were exposed to 18:2, revealed noreduction in lipid-like droplet accumulation in compar

ison to cells treated with 18:2 alone. It is possible thatvitamin E reduces the 18:2-induced increase in peroxisomal ß-oxidationby formation of vitamin E complexes

with free fatty acids (29), possibly by formation of ahydrogen bond between the a-tocopherol chromanol

nucleus hydroxyl and the carboxyl group of a fatty acid(31). Vitamin E may thus prevent the induction of peroxisomal enzymes by blocking the carboxyl end of thefatty acid, an action that may prevent peroxisomal ß-ox

idation of fatty acids as well as excess formation ofcytotoxic hydrogen peroxide, a by-product of peroxisomal ß-oxidation. Since ß-oxidationcould be impededby vitamin E, cellular lipid degradation might be reduced. In this event, a decrease in cytoplasmic lipid-like

droplets should not be expected.In summary, our data suggest that the protective

effects of vitamin E against fatty acid-mediated endo

thelial cell injury may be due in part to the ability ofvitamin E to reduce peroxisomal ß-oxidationas a consequence of decreased fatty acid-mediated induction of

peroxisomes. These protective properties of vitamin Eagainst endothelial cell injury may have implications inregard to understanding the etiology of atherosclerosis.Further studies are needed to clearly define the relationship between peroxisome proliferation and atherosclerosis.

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