ANTONIO MARTIN,* DAYONG WU,* WENDY BAUR, ~ SIMIN N. MEYDANI,* JEFFREY B. BLUMBERG,* and MOHSEN MEYDANI *
• Jean Mayer USDA Human Nutrition Research Center on Aging at Tufts University, Boston, MA, USA; and tNew England Medical Center, Boston, MA, USA
(Received 21 July 1995; Revised 27 December 1995; Accepted 29 February 1996)
Abstract--Reactive oxygen species produced by the cells present in the arterial wall may cause oxidative damage to cellular components altering endothelial cell (EC) function. Changes in the EC function appear to play a key role in the pathogenesis of atherosclerosis. Human aortic endothelial cells (HAEC) were employed to investigate the protective role of vitamin E upon exposure of endothelial cells to oxidative stress in vitro. HAEC assimilate d-a- tocopherol from the media in a dose-dependent manner. Exposure of HAEC to 16.5 mM of the free radical generator 2,2'-azobis (2-amidinopropane) hydrochloride (AAPH) for 16 h decreased cell viability (assessed by trypan blue exclusion) from 90 to 28%. HAEC preincubated with vitamin E at 15, 30, and 60 #M prior to the AAPH exposure resulted in a dose-dependent increase in resistance to oxidative stress and increased cell viability by 37, 66, and 85%, respectively. An increase in prostacyclin (PGI2) production by HAEC in response to AAPH exposure was correlated positively with cell damage and negatively with vitamin E concentration. Interleukin (IL)-1 production also increased in parallel with cell damage induced by AAPH. Vitamin E treatment significantly reduced IL-1 production after AAPH exposure. This modulatory role of vitamin E on HAEC function following exposure to an oxidative stress may reflect its antioxidant protection against lipid peroxidation.
Keywords--Vitamin E, Oxidative injury, Prostacyclin (PGI2), Interleukin (IL-1), Human aortic endothelial cells, free radicals
INTRODUCTION
Despi te s ignif icant efforts to unders tand the events that lead to the fo rmat ion o f a the romatous les ions , the changes that cause reg ions of the ar ter ia l wal l to be- come " l e s i o n p r o n e " are not wel l unders tood. There is ev idence that in jury to arterial endo the l i a l cel ls (EC) represent one o f the key events in the ini t ia t ion and evo lu t ion o f a therosc leros is . ~ The endo the l ium is a mul t i func t iona l t issue that ac t ive ly par t ic ipa tes in the ma in tenance o f ca rd iovascu la r homeos ta s i s .2 Vascu la r EC interact d i rec t ly with cel ls and molecu le s f rom the b lood and under ly ing t issues. M a n y o f the in terac t ions
Address correspondence to: Mohsen Meydani, DVM, Ph.D., JM, USDA Human Nutrition Research Center on Aging, at Tufts Uni- versity, 711 Washington Street, Boston, MA 02111.
Current address of Antonio Martin: Whitaker Cardiovascular In- stitute, Boston University School of Medicine, 80 East Concord Street, Boston, MA 02118.
o f EC with cel ls such as neut rophi l s and macrophages appear to invo lve the EC as targets o f ox ida t ive injury. In response to ox ida t ive stress, EC can p roduce and re lease supe rox ide (O1-), hyd rogen pe rox ide (H202), and cy tok ines l ike in ter leukin (IL)-1 and e icosano ids l ike p ros tacyc l in (PGIz). Reac t ive oxygen species (ROS) are also p roduced by red b lood cel ls 3 and cel ls invo lved in in f l ammatory process l ike neut rophi l s and monocy te s 4 as wel l as cel ls f rom the vessel wal l in- c lud ing smooth musc le cel ls and saphenous EC. 5'6 ROS can cause ox ida t ive d a m a g e to ce l lu la r compo- nents and loss o f EC f u n c t i o n ] Changes in EC m e m b r a n e funct ion induced by ROS and l ip id per- ox ida t ion appear to p l ay a key role in the pa thogenes i s o f a therosc leros is . 1
ROS appear to have impor tant roles in several vas- cular processes including atherosclerosis . It has been shown that d ie ta ry- induced hypercholes te ro lemia is
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followed almost immediately by an increased adhesion of monocytes and neutrophils to EC and an increased release of O~ .8 In addition, nitric oxide (NO'), a com- pound involved in a wide variety of physiologic and pathologic functions, is also produced by vascular EC and is highly reactive with O2- and produces peroxy- nitrate (ONOO-). Peroxynitrate can act as a pro-oxi- dant and cause cell injury. 9
IL-1 has been implicated in the pathogenesis of ath- erosclerosis. 1° In response to oxidative stress EC pro- duce IL-1, which stimulate EC release of chemotactic factors and increase the expression of cell surface ad- hesion molecules to neutrophils, monocytes, and lym- phocytes] 1 PGI2, also secreted by EC, has antiaggre- gatory and vasodilatory properties] 2 In addition, PGI2 has been implicated as a cytoprotectant acting to di- minishing ROS production by neutrophils and release of lysosomal enzymes] 3 PGI2 is also released when EC are stressed by including mechanical shearing forces or oxidative injury. 14
Epidemiological and clinical studies have indi- cated that dietary vitamin E reduces the risk of car- diovascular disease. ~5 In vitro studies have shown that vi tamin E protects EC from oxidative stress dam- age.16 Vitamin E is located in the lipid bilayer of the membrane and protects both the membrane and membrane-bound enzymes from oxidative stress, j7 In addition to its antioxidant function, vitamin E ap- pears to form stable physicochemical complexes within the membrane lipid bilayer influencing pro- cesses such as membrane receptor insertion and movement of receptor-l igand complexes, ~s and mod- ulating eicosanoid and cytokine production. 19'2° Vita- min E and synthetic antioxidants such as probucol and butylated hydroxytoluene have been shown to slow the development of atherosclerotic lesions in nonhuman primates and hypercholesterolemic rab- bits and hamsters. 2~-24 However , in contrast to syn- thetic antioxidants, dietary vitamin E is readily in- corporated into biological membranes and has no toxic effects. 25'26 Recently, Hodis et al. 27 reported a significant association between vitamin E supple- mentation and angiographically demonstrated reduc- tion in coronary artery lesion progression in patients with coronary artery bypass grafts.
Thus, we used a human aortic EC (HAEC) culture system to evaluate the effect of vitamin E against oxi- dative stress induced by ROS. We used the water-sol- uble free radical generator 2,2'-azobis (2-amidinopro- pane) hydrochloride (AAPH) to simulate the in vivo conditions of oxidative stress. AAPH undergoes spon- taneous thermal decomposition producing carbon-cen- tered radicals at a constant rate. 28 In the presence of oxygen, these radicals can attack membrane polyun-
saturated fatty acids and initiate lipid peroxidation chain reactions.
MATERIALS AND METHODS
Endothelial cell culture
HAEC were purchased from Clonetics Laboratories (San Diego, CA) and cultured in M-199 medium with- out phenol red (Gibco, Grand Island, NY). Culture me- dium contained 10% fetal bovine serum (FBS) (Sigma Chemical Co., St. Louis, MO), 5 #g/ml EC-derived growth factor prepared from bovine retina, 100 #g/ml EDTA, 100 U/ml penicillin, 100 U/ml streptomycin, and 1.25 #g/ml amphotericin B (Sigma Chemical Co.). HAEC were cultured on 1% gelatin-coated flasks in six-well Coming plates (Corning, NY). The medium was replaced every 2 d until the cells attained conflu- ence. Fourth to seventh passage cells were employed, and all experiments conducted 24 h after HAEC reached confluence using six wells per experimental treatment. Under inverted microscopy (Zeiss, West Germany) confluent HAEC monolayers displayed a cobblestone phenotype typical of quiescent EC and were characterized by the presence of yon Willebrand factor antigen using immunofluorescent microscopy. 29 Cells were grown to confluence in 20% 02, 5% CO2, and balance N2. Following treatment of HAEC with 0.05% trypsin for 3 min or until 80% of the cells were detached, cell viability was determined by trypan blue exclusion test and expressed by the percent of cells that excluded the dye.
Confluent HAEC from fifth passage grown in six- well plates (Coming, Corning, NY) were supplemented with a-tocopherol by incubating them in M-199 me- dium containing 0, 15, 30, and 60 #M a-tocopherol for 24 h. Following supplementation, supernatant was re- moved, HAEC washed with HANKS solution, and cul- ture media replaced with new media containing 16.5 mM AAPH and the cells incubated for 16 h. AAPH was prepared by dissolving 60 mg AAPH in 15 ml of M-199 media. After HAEC exposure to AAPH the su- pernatant was collected and stored at -80°C for later determination of PGI2 and IL-1/3.
Vitamin E supplementation
A stock solution of d-c~-tocopherol (Kodak, Spring- field, NJ) containing 10 mg/ml was prepared in abso- lute ethanol and stored at -70°C. To supplement the culture media with vitamin E, the required amount of d-c~-tocopherol from stock solution was dried under ni- trogen and redissolved with ethanol to achieve a final concentration of 0.05% ethanol in the culture media.
Effect of vitamin E on human aortic endothelial cell responses 507
The a- tocopherol-e thanol solution was mixed with I0% FBS, then incubated at 37°C for 15 min, while mixing gently every 5 min. The HAEC were incubated for 22 h in media containing various concentrations of a-tocopherol incorporated into the FBS.
Vitamin E determination
Concentration of vitamin E in HAEC and media was measured by high-performance liquid chromatography (HPLC). Briefly, tocol (gift from H o f f m a n n - L a Roche, Nutley, NJ) was added to HAEC cells or media as an internal standard. Following extraction with hex- ane, the sample was dried under nitrogen and reconsti- tuted in methanol. Tocopherol peaks were separated using a C18 reverse phase column packed with 3 # particles using 100% methanol as a mobile phase. 6 Eluted peaks were detected by a Perk in-Elmer 650-15 fluorescence spectrophotometer (Norwalk, CT) set at 292 nm excitation and 330 nm emission wave lengths. Peaks were integrated with a Waters 860 system (Mil- ford, MA).
PGI2 and IL- l fl
After HAEC were incubated with AAPH for 16 h, 200 #1 aliquots of media were collected and stored at -80°C. PGI2 was analyzed by radioimmunoassay (RIA) for 6-keto-PGG~, the main hydration product of PGI2 as described by McCosh et al. 3° IL-1 secreted into the media was measured by RIA according to the method of Endres et al. 31
Statistical analysis
Significant differences in the measured parameters between treated and control cells were calculated using Student's t-test at p < .05. Values are presented as mean + SD.
RESULTS
Exposure of HAEC to AAPH resulted in marked cell damage and loss of cell viability (Fig. 1). Cell viability decreased to 28 ___ 3% compared to control (see second bar vs. first bar in Fig. l). We have previously shown that incubation of HAEC with medium containing c~- tocopherol up to 54 #M for 22 h increased cellular c~- tocopherol content in a dose-dependent fashion. 6 Thus, supplementation of HAEC with c~-tocopherol prior to the addition of AAPH protected the cells from oxida- tive damage and increased cell viability in a dose-de- pendent fashion. HAEC preenriched with c~-tocopherol in the medium at concentrations of 15, 30, and 60 #M
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5 0
~ 40.
30"
20"
10
A A P H (mM)
- - r -
T
T
0 0 15 30 60 0 16.5 16.5 16.5 16.5
Fig. 1. Cell viability of HAEC following exposure to AAPH. Con- fluent HAEC presupplemented without a-tocopherol and not exposed to AAPH (control), or with different concentrations of a-tocopherol and exposed to 16.5 mM AAPH were incubated for 16 h. Each bar represents the mean _+ SD of six wells per treatment, a-T = a- tocopherol. *Significantly different from control (p < .05).
increased cell viability to 37 + 8%, 66 + 4%, and 85 + 5%, respectively, compared to control HAEC (90 + 1%) without AAPH exposure (Fig. 1). The increase in cell viability was directly proportional to the concen- tration of c~-tocopherol in the HAEC (r = .99, p < .001).
When confluent HAEC were supplemented with 0, 15, 30, and 60 #M c~-tocopherol in the medium for 22 h and then exposed to AAPH-induced oxidative stress, the concentration of c~-tocopherol in the unsupple- mented, treated cells was lower than control cells (0.09 + 0.02 vs. 0.03 _+ 0.01 nmol/106 cells, p < .05). (Fig. 2). The concentration of a-tocopherol in the HAEC af- ter AAPH exposure was proportional to their presup- plementation concentrations in the medium (Fig. 2). HAEC that were exposed to AAPH, but preincubated with 15, 30, and 60 #M c~-tocopherol had 6-, 13-, and 33-fold higher c~-tocopherol concentrations, respec- tively, compared to control, which had only 0.09 nmol/ 106 cells.
Effect of ol-tocopherol and AAPH on PG12 production by HAEC
HAEC, incubated with 16.5 mM AAPH, had a five- fold increase in PGI2 release compared to control cells
508 A. MARTIN et al.
j
O
8
4 . 0 '
3.5'
3.0'
2.5'
2.0'
1.5'
1.0
0.5"
0.0 ~-T 1 tM) 0
A A P H (riM) 0
* I .';';';';';'
0 60 16.5 16.5
15 30 16.5 16.5
--Y- ,~,~.~,~,~.~
,.~,-,-.-.;,
'~l~/%lgg%g k
Fig. 2. Concentration of a-tocopherol in HAEC following exposure to AAPH. Confluent HAEC were presupplemented without o~-to- copherol and not exposed to AAPH (control) or with different con- centrations of a-tocopherol for 24 h before exposure to 16.5 mM AAPH for 16 h. Each bar represents the mean _+ SD of six wells per treatment, a-T = a-tocopherol. Significantly different from con- trol *p < .05, **p < .01.
(129 + 7 vs. 751 +__ 172 pg/ml) (Fig. 3). AAPH-induced production of PGI2 by HAEC decreased as the cells were pretreated with increasing concentrations of a- tocopherol. AAPH increased PGI2 production in un- supplemented and in all supplemented HAEC. Enrich- ing HAEC with c~-tocopherol without inducing oxidative stress by AAPH, only slightly increased PGI2 production (data not shown). The production of PGI2 by HAEC was directly related to the magnitude of cell damage (assessed by trypan blue exclusion) (Fig. 4) and inversely associated with the concentration of a- tocopherol (r = .83, p < .001). HAEC, supplemented with the highest concentration of c~-tocopherol (60 #M) relative to other vitamin E-supplemented and -unsupplemented EC, showed the greatest protection against injury and the smallest PGI2 response (p < .05). (Fig. 3)
Effect of ol-tocopherol and AAPFI on IL-113 production by HAEC
Production of IL-1/3 by HAEC was significantly higher in the cells exposed to AAPH compared to con- trol cells (Fig. 5). IL- lp secretion by the control HAEC was 204 _.T_ 16 pg/ml/106 cells and enrichment with dif- ferent levels of a-tocopherol without induction of ox- idative stress with AAPH had no effect on IL-I/3 pro-
1000"
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70¢
600
500
400
300
200
100
ot-T (p-M) 0 0 15 30 60 A A P H (nM) 0 16.5 16.5 16.5 16.5
Fig. 3. PGIz production by HAEC following exposure to AAPH. Confluent HAEC were presupplemented without a-tocopherol and not exposed to AAPH (control) and with different concentrations of a-tocopherol for 24 h then exposed to 16.5 mM AAPH for 16 h. Each bar represents the mean _+ SD of six wells per treatment, c~- T = c~-tocopherol. Significantly different from control *p < .05, **p < .01.
duction (data not shown). However, following AAPH oxidative stress, its production increased by 4.4-fold to 895 +_ 75 pg/ml/106 cells (p < .005) in unsupplemented cells. AAPH increased IL-1/3 production in all the HAEC enriched with c~-tocopherol. IL-1/3 production
1000"
800'
600"
~ 400"
200'
/ • R = 0 . 8 m ~ 9 a / ~ a p<0.001
o " 2 ' 0 g0 " 6 . 0 8'0 Cell Damage (% of cel ls stained)
Fig. 4. Correlation of PGI2 production with cell damage and a- tocopherol concentration in HAEC. The PGI2 released by HAEC following exposure to 16.5 mM AAPH is plotted against percent nonviable cells in culture.
Effect of vitamin E on human aortic endothelial cell responses 509
Fig. 5. IL-1/3 production by HAEC following exposure to AAPH. Confluent HAEC were supplemented without c~-tocopherol and not exposed to AAPH (control) and with different concentrations of a- tocopherol for 24 h then exposed to 16.5 mM AAPH for 16 h. Each bar represents the mean _+ SD of six wells per treatment, ot-T = ot- tocopherol. Significantly different from control. *p < .005.
was most markedly suppressed (403 _ 129 pg/ml/106 cells) (p < .05) with 60 #M c~-tocopherol. The increase in IL- 1/3 secretion was directly related to the magnitude of the cell injury (Fig. 6) and inversely associated with the concentration of a-tocopherol in the HAEC (r = .85, p < .001).
DISCUSSION
Enrichment of HAEC with c~-tocopherol in vitro provided protection against oxidative injury induced by AAPH and decreased PGI2 and IL-1/3 production by the cells. The degree of HAEC damage was propor- tional to the a-tocopherol concentration of the cells. The lowest vitamin E dose (15 #M) is roughly equiv- alent to the plasma a-tocopherol concentrations found in healthy persons consuming a diet low but not defi- cient in vitamin E. 32 The highest dose (60 #M) is sim- ilar to the plasma level of c~-tocopherol achieved with regular vitamin E supplementation at about 200 IU. 25
Vitamin E has been shown to protect against free radical-induced oxidative damage in several in vitro cell models including EC from umbilical vein, 33 por- cine pulmonary artery, 16 rat heart muscle, 34 and rat aor- tic smooth muscle. 35 Unlike EC from vein and bovine aorta, HAEC may be a better model for the study of atherogenesis, as they possess such relevant character- istics as: production of endothelium-derived relaxing factor, H202 and prostaglandins, maintenance of inter-
cellular junctions, and the activity of certain enzymes such as superoxide dismutase and catalase. 6'36-37
PGI2 is produced by EC and possesses antiaggre- gatory and vasodilatory properties and decreases poly- morphonuclear cell (PMN) adhesion to EC in vitro. 38 Under physiological conditions, the secretion of PGI2 by human endothelial cells in vivo and under normal EC culture conditions is extremely low. 39 However, production of PGI2 increases under conditions associ- ated with atherosclerosis and platelet activation. 39-41 Histamine release and mechanical stresses such as the shear stress of blood flow have also been reported to increase PGI2 production by EC. 42 This PGI2 response to oxidative and physical stress may be important to the maintenance of vascular homeostasis. This concept is supported by studies using drugs 4°'43 and oxidized low-density lipoprotein. 44'45 Production of PGI2 by HAEC was found to be directly proportional to the ex- tent of AAPH-induced oxidative stress and cell damage (Fig. 3). When the cells were enriched with a-tocoph- erol, the oxidative stress and resultant injury were sig- nificantly diminished, and PGI2 production was less than that of unsupplemented cells.
Several cytokines have been implicated in normal and pathogenic vascular functions through their auto- crine and/or paracrine effects.11.46 Production of IL- 1/3 by EC under normal conditions is very low. 47"48 HOW- ever, in response to oxidative stress and injury, the pro- duction of IL-1/3 increases and augments the expres- sion of intercellular adhesion molecule-1 (ICAM- 1) and vascular adhesion molecule-1 (VCAM-1) by EC, thereby increasing adhesion of circulating monocytes and lymphocytes to the activated EC at the site of in- j ury.ll.49 IL-1/3 also acts to regulate the production of
I000-
80O
,oo
200'
p<O.O01
Cel l d a m a g e (% of ce l l s s tained)
Fig. 6. Correlation of IL-1/3 production by HAEC with cell damage and ot-tocopherol concentration. Amount of IL- 1/3 released by HAEC following exposure to 16.5 mM AAPH is plotted against percent of nonviable cells in culture.
510 A. MARTIN et al.
chemokines such as monocyte chemoattractant protein- 1 (MCP-1) by macrophages. 5° Thus, IL-1/3 secretion by vascular cells appears to be intimately involved in monocyte recruitment and adhesion, which, together with the production of other cytokines and growth fac- tors, are crucial events in the initiation and progression of the atheromatous plaque. 5~-53
The oxidative stress induced by AAPH resulted in secretion of IL-1/3 by HAEC in parallel with cell dam- age (Fig. 5). This response may be related to the early stages of atherosclerosis where the expression of cy- tokine-induced leukocyte adhesion molecules is an im- portant component of recruiting leukocytes to the le- sion area. The enrichment of HAEC with a-tocopherol protected them against the oxidative damage and de- creased IL-1/3 production. It is most likely that the an- tioxidant function of a-tocopherol in the membrane of supplemented cells was mainly responsible for block- ing free radical damage to lipids and proteins in the cell membrane when exposed to free radicals produced by AAPH. However, the observed effect may not be solely mediated by the antioxidant function of a-tocopherol.
In summary, vitamin E supplementation reduced AAPH-induced cell damage and PGI2 and IL-1/3 pro- duction. Thus, this effect of vitamin E on prostanoid and cytokine production, in addition to its inhibition of the oxidative modification of low-density lipoprotein, may contribute to the previously reported effect of vita- min E in reducing the risk of cardiovascular diseases.
Acknowledgements - - The authors would like to thank Ms. Jennifer Munnis for her assistance in preparation of the manuscript. This pro- ject was supported with federal funds from the U.S. Department of Agriculture, Agricultural Research Service under contract number 53-3K06-0-1. The content of this publication does not necessarily reflect the policy of the U.S. Department of Agriculture, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government.
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