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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online November 3, 2003 as doi:10.1096/fj.03-0301fje. |
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Department of Internal Medicine III, Division of Endocrinology and Metabolism, University of Vienna, A-1090 Vienna, Austria
2Correspondence: Department of Internal Medicine III, Division of Endocrinology and Metabolism, Waehringer Guertel 18-20, A-1090 Vienna, Austria. E-mail: sabina.baumgartner-parzer{at}akh-wien.ac.at
SPECIFIC AIMS
Plasma concentrations of free fatty acids (FFAs) are increased in states relating to the metabolic syndrome and impair endothelial function. To characterize the effects of FFAs on vascular cell function, human umbilical vein endothelial cells (HUVECs) exposed to selected purified (saturated, monounsaturated, and polyunsaturated) FFAs and to elevated plasma FFA concentrations, as induced in humans, were studied with respect to 1) apoptosis, 2) cell cycle progression, and 3) associated gene/protein expression.
PRINCIPAL FINDINGS
1. Selected purified FFAs saturation-dependently modulate endothelial apoptosis
Stearic acid (C18:0; 24 h), a saturated fatty acid (SFA), concentration-dependently increased (Fig. 1
A) whereas oleic acid (C18:1
9), a monounsaturated fatty acid, did not affect apoptosis in HUVECs (Fig. 1B
). The polyunsaturated fatty acids (PUFAs) linoleic (C18:26; Fig. 1C
), 
-linolenic (C18:36; Fig. 1D
), and arachidonic acid (C20:46; Fig. 1E
) induced endothelial apoptosis only at the highest concentration used (300 µmol/L). Extending exposure time to the FFAs (300 µmol/L) from 24 h (hatched/gray bars) to 48 h (black bars) further increased endothelial apoptosis, which was then also triggered by oleic acid (Fig. 1F
). The apoptotic activity of oleic acid and PUFAs increased (P<0.001) with the number of double bonds (24 h: r=0.75; 48 h: r=0.66).
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2. BSA-conjugated linoleic acid
BSA-conjugated linoleic acid (C18:26; 24 h, 300 µmol/L) had proapoptotic activity similar to unconjugated linoleic acid as revealed by a 200% rise of apoptosis in HUVECs vs. control cells exposed to 150 µmol/L BSA (set to 100%). BSA (150 µmol/L) per se did not affect apoptosis in HUVECs.
3. FFA-induced endothelial apoptosis relates to bak expression
In HUVECs (n=5), protein expression of the apoptosis promotor bak was concentration-dependently increased by stearic acid (C18:0; Fig. 2
) but not by oleic acid (C18:19). PUFAs (C18:26, C18:36, C20:46) induced endothelial up-regulation of bak only at concentrations of 300 µmol/L (Fig. 2)
. Proapoptotic activity of stearic acid (r=0.99, P<0.01) and unsaturated FFAs (r=0.96, P<0.05) correlated with endothelial protein expression of bak; all FFAs tested reduced protein expression of the apoptosis inhibitor bcl-2 (–23±3%; P<0.001; n=25; n=5 for each FFA) independent of their respective apoptotic activity.
4. Selected purified FFAs modulate cell cycle progression differently
Stearic acid (C18:0; 300 µmol/L, 48 h) increased the number of HUVECs in G0/G1 phase of the cell cycle (+14±5%; n=6, P<0.05) and reduced that of S phase cells (–15±4%; P<0.05) without affecting the number of cells in G2/M phase vs. intraindividual ethanol-treated cells as control. Whereas oleic acid (C18:19) only marginally diminished the number of S phase cells (–4±1%, P<0.05), PUFAs (300 µmol/L) caused a marked G0/G1 cell cycle arrest (C18:26: +33±12%, C18:36: +32±11%, C20:46: +26±9%; P<0.001) by reducing the numbers of cells in S (C18:26: –22±11%, P<0.05; C18:36: –22±10%, P<0.01; C20:46: –19±10%, P<0.01) and G2/M phases (C18:26: –11±2%, P<0.001; C18:36: –9±2%, P<0.001; C20:46: –8±4%; P<0.01).
5. FFA-induced cell cycle arrest relates to p21WAF-1/Cip1 expression
Independent of their ability to delay cell cycle progression, all FFAs reduced endothelial cyclin D3 protein expression by 36 ± 3% (P<0.001, n=25; n=5 for each FFA).
300 µmol/L stearic acid (C18:0: +34±6, P<0.01) and PUFAs (C18:26: +72±36%, C18:36: +87±27%, C20:46: +40±12%; P<0.05), but not oleic acid (C18:19), increased protein expression of the inhibitor of cyclin-dependent kinases p21WAF-1/Cip1 in HUVECs (n=5), exhibiting a close relationship (r=0.94, P<0.01) with the number of cells residing in the G0/G1 phase of the cell cycle. However, FFAs did not affect protein expression of p53.
6. Selected purified FFAs alter secretory profile of endothelial cells
All FFAs concentration-dependently reduced (P<0.001) expression of NF-
B’s inhibitor, IB
(–50±5%), the multifunctional molecule clusterin (–54±5%), and the vasoactive molecules endothelial NO synthase (eNOS; –44±4%) and pre-proendothelin-1 (ppET-1; –44±5%) in HUVECs (n=25; n=5 for each FFA).
7. High plasma FFA concentrations trigger endothelial apoptosis
High plasma FFAs induced in volunteers by infusion of intralipid (mean FFA concentration: 369±24µmol/L, n=4) or heparin (FFA concentration: 387 µmol/L, n=1) induced a 1.9- (P<0.01) and 4.2-fold (P<0.001) increase of apoptosis in HUVECs (n=9) after 24 and 48 h exposure, respectively, compared with cells treated with the intraindividual control samples containing low FFAs (56±21µmol/L) obtained before Intralipid or heparin infusion.
CONCLUSIONS AND SIGNIFICANCE
Lifestyle-associated disorders such as the metabolic syndrome, including hypertension, obesity, and diabetes, are associated with increased plasma concentrations of FFAs.
This study demonstrates induction by selected purified fatty acids of apoptosis in human vascular endothelial cells. The observation of plasma FFA concentrations elevated in humans in vivo by lipid or heparin infusion to also trigger endothelial apoptosis supports the contention for a detrimental potential for the endothelium by FFA stimulation in vivo. Studies of the effects of selected purified FFAs revealed that the proapoptotic action of stearic acid is concentration dependent (100–300 µmol/L), whereas endothelial apoptosis induced by polyunsaturated FFAs (PUFAs) increases with the number of fatty acid double bonds.
FFA proapoptotic activities correlated with protein expression of the apoptosis promotor bak and were associated with a p21WAF-1/Cip1-related G0/G1 cell cycle arrest. All FFAs reduced the expression of IB (a marker of NF-B activation) to a similar extent, suggesting that NF-B does not play a major role in FFA-mediated endothelial cell death. This also holds true for the atheroprotector clusterin, and the vasoactive molecules endothelin-1 and endothelial NO synthase.
In contrast to stearic acid and PUFAs, the monounsaturated oleic acid triggered neither apoptosis nor G0/G1 cell cycle arrest. These findings are in line with the idea that a high intake of saturated and polyunsaturated fatty acids contributes to, whereas oleic acid, the predominant fatty acid in olive oil and the Mediterranean diet, attenuates the development of atherosclerosis.
The concentrations of stearic, oleic, and linoleic acid used in this study (100–300 µmol/L) are within the physiologic concentration range. Under pathophysiologic conditions, systemic plasma FFA concentrations can rise to the millimolar range; endothelial cells in particular might be locally exposed to excessively high FFA concentrations due to lipolysis of lipoprotein triglycerides by the endothelium-bound lipoprotein lipase.
Our data show that a physiologic rise in plasma FFA directly induces vascular dysfunction by triggering endothelial cell apoptosis. Recent evidence suggests that endothelial cells undergoing apoptosis impair the endothelial barrier function, trigger atherosclerotic plaque erosion and plaque rupture, exert proadhesive and procoagulatory activity, and represent key regulators of endothelial dysfunction and overt micro- and macrovascular disease, including atherosclerosis and diabetic retinopathy.
The finding that saturated and polyunsaturated (6) FFAs, but not oleic acid, inhibit cell cycle progression and trigger apoptosis suggests that FFA structure affects endothelial cell function differently. By such action, FFAs could differently modulate endothelial cell dysfunction which reflects very early stages of micro- and macrovascular disease.
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FOOTNOTES
1 To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.03-0301fje 
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