Aspirin protects human coronary artery endothelial cells against
atherogenic electronegative LDL via an epigenetic mechanism: a novel
cytoprotective role of aspirin in acutemyocardial infarction
Po-Yuan Chang1, Yi-Jie Chen2, Fu-Hsiung Chang2, Jonathan Lu3, Wen-Huei Huang2,
Tzu-Ching Yang2, Yuan-Teh Lee1, Shwu-Fen Chang4, Shao-Chun Lu2*, and Chu-Huang
Chen3,5,6,7*
1. Department of Internal Medicine, National Taiwan University College of Medicine,
Taipei, Taiwan; 2. Department of Biochemistry and Molecular Biology, National
Taiwan University College of Medicine, No. 1, Sec. 1, Jen-Ai Road, Taipei 100, Taiwan;
3. Vascular and Medicinal Research, Texas Heart Institute at St. Luke’s Episcopal
Hospital, 6770 Bertner Avenue, MC 2-255, Houston, TX 77030, USA; 4. Graduate
Institute of Cell and Molecular Biology, Taipei Medical University, Taipei, Taiwan; 5.
Department of Medicine, Baylor College of Medicine, Houston, TX, USA; 6. Graduate
Institute of Clinical Medical Science, China Medical University, Taichung, Taiwan; and
7. L5 Research Center, China Medical University Hospital, Taichung, Taiwan Received
24 July 2012; revised 11 February 2013; accepted 11 March 2013; online
publish-ahead-of-print 20 March 2013
Time for primary review 22 days
Aims
L5 is the most negatively charged subfraction of human low-density lipoprotein (LDL)
and is the only subfraction of LDL capable of inducing apoptosis in cultured vascular
endothelial cells (ECs) by inhibiting fibroblast growth factor-2 (FGF2) transcription.
We examined whether plasma L5 levels are elevated in patients with ST-segment
elevation myocardial infarction (STEMI) and whether aspirin provides epigenetic
protection of human coronary artery ECs (HCAECs) exposed to L5.
Methods and results
Plasma L5 levels were compared between patients with STEMI (n ¼ 10) and control
subjects with chest pain syndrome but a normal coronary arteriogram (n ¼ 5). L5 was
isolated from the plasma of STEMI patients and control subjects, and apoptosis, FGF2
expression, and FGF2 promoter methylation were examined in HCAECs treated with
L5 and aspirin. Plasma L5 levels were significantly higher in STEMI patients than in
control subjects (P , 0.001). Treatment of HCAECs with L5 resulted in reduced survival
and FGF2 expression and increased CpG methylation of the FGF2 promoter.
Co-treatment of HCAECs with L5 and a physiologically relevant, low concentration of
aspirin (0.2 mM) attenuated the adverse effects of L5 on HCAEC survival, FGF2
expression, and FGF2 promoter methylation. In contrast, high concentrations of
aspirin (≥1.0 mM) accentuated the effects of L5.
Conclusions
Our results show that L5 levels are significantly increased in STEMI patients.
Furthermore, L5 impairs HCAEC function through CpG methylation of the FGF2
promoter, which is suppressed in the presence of low-concentration aspirin. Our
results provide evidence of a novel mechanism of aspirin in the prevention of MI.
Keywords
Aspirin: DNA methylation; Genes; Lipoproteins; Myocardial infarction
1. Introduction
The antiplatelet effect of aspirin (acetylsalicylic acid) that results from its inhibition
of cyclooxygenase enzymes has been well described and is the pharmacologic basis
of aspirin use in the prevention of acute myocardial infarction (MI), including
ST-segment elevation MI (STEMI) and non-STEMI.1,2 Aspirin has also been shown to
have cytoprotective functions that are unrelated to its antiplatelet activity,3,4 such
as improving endothelium-dependent arterial relaxation by inducing the release of
nitric oxide from the vascular endothelium5 and reducing apoA levels in human
hepatocytes by suppressing apoA gene transcription.6 Thus, aspirin may protect
against MI through mechanisms independent of its antiplatelet activity.
Electronegative low-density lipoprotein (LDL) is a class of naturally occurring
atherogenic lipoproteins.7 Human plasma can be chromatographically resolved into
five subfractions, L1–L5, with increasingly negative charge.8,9 L5, the most
electronegative subfraction, is the only subfraction capable of inducing marked
atherogenic changes in cultured vascular endothelial cells (ECs) and is moderately
elevated (up to 5–6% of LDL) in asymptomatic individuals with increased cardiac risks,
including hypercholesterolaemia, active smoking, type 2 diabetes, and metabolic
syndrome.8,10 – 13 L5 differs from experimentally derived oxidized LDL (oxLDL)
physically and chemically,11,14 but both forms of LDL share similar functional
properties and signal through the lectin-like oxLDL receptor-1 (LOX-1).15 – 17 In
cultured vascular ECs, L5 and oxLDL induce apoptosis by inhibiting the expression of
fibroblast growth factor-2 (FGF2),8,18,19 a pleiotropic protein that maintains normal
endothelial physiology20 and protects against MI.21,22 We have previously shown
that homocysteine, another protein that may be linked to cardiovascular disease,
suppresses FGF2 transcription through CpG methylation of the FGF2 promoter.23
However, it is not known whether CpG methylation of the FGF2 promoter is
inducible by atherogenic LDL.
In our study, we hypothesized that L5 levels are elevated in the plasma of STEMI
patients. In addition, we examined in human coronary artery ECs (HCAECs) whether
aspirin attenuates the effects of L5 on cell survival and FGF2 expression and whether
CpG methylation is involved. Our results provide evidence of a novel protective
mechanism of low-dose aspirin independent of its antiplatelet activity and
substantiate the importance of aspirin therapy for protecting against MI.
2. Methods
2.1 Study subjects
This study was approved by the institutional review board of the National Taiwan
University Hospital and conforms with the principles outlined in the Declaration of
Helsinki. All participants gave written informed consent. We analysed 10 subjects
with STEMI undergoing primary coronary intervention and 5 control subjects with
chest pain syndrome but a normal coronary arteriogram. The criteria for STEMI were
defined
according to the consensus definition of the American Heart Association/American
College of Cardiology.24
2.2 Cell culture
HCAECs (Clonetics, Lonza Group Ltd., Switzerland) were maintained in EGM-MV
medium supplemented with 20% foetal bovine serum and antibiotics (Clonetics). Cell
cultures (passages 4–7) grown to subconfluence were washed three times with the
serum-free medium and were maintained under serum-free conditions for 6 h
before being treated with various agents according to the protocols determined by
preliminary experiments. Eighteen hours after the designated treatment, cells were
incubated with phosphate-buffered saline (PBS; lipoprotein-free control), L1, or
STEMI L5 (50 mg/mL each) for 24 h in the presence or absence of aspirin (0–5 mM).
At least three independent experiments (each in triplicate) were performed for each
treatment group.19,23
2.3 LDL preparations
Plasma LDL obtained from STEMI patients and control subjects was resolved into
subfractions L1–L5 by fast protein liquid chromatography equipped with an
anion-exchange column, as described previously.8,9 OxLDL was prepared by
copper oxidation of L1 from control subjects as described previously.18,19 Control
LDL was L1 from control subjects. Protein concentration was estimated by the Lowry
method. Agarose gel electrophoresis of LDL preparations was performed by using
the Beckman Paragon system (Beckman, Palo Alto, CA, USA).
2.4 Analysis of FGF2 protein levels and DNA synthesis
HCAECs (1 × 106 cells/well) were seeded in 12-well Corning cell culture plates
(Corning, Lowell, MA, USA) and treated with increasing concentrations of aspirin (0,
0.2, 1, 3, or 5 mM) in the presence of PBS, L5 from STEMI patients (STEMI L5; 50
mg/mL), or oxLDL (50 mg/mL) for 24 h. DNA synthesis was quantified by measuring
3H-thymidine (Moravek Biomedicals, Brea, CA, USA) incorporation.18,19 FGF2
protein concentrations were measured with an enzyme-linked immunosorbent assay
(ELISA) by using a Quantikine kit (R&D Systems, Minneapolis, MN, USA).
2.5 Internalization of L5 by HCAECs
The internalization of L5 into HCAECs was studied by using L5 labelled with
1,1′-dioctadecyl-3,3,3′,3′
tetramethylindocarbocyamine
perchlorate
(DiI;
Sigma-Aldrich, St Louis, MO, USA).25 HCAECs were pre-treated with aspirin (0.2 mM),
anti-LDL receptor (LDLR; 0.1 mg/mL; R&D Systems), or anti-LOX-1 (0.1 mg/mL; R&D
Systems) at 378C for 16 h. Cells were washed with the fresh medium, followed by
incubation with 50 mg/mL DiI-labelled L5 at room temperature for 1 h. Intracellular
DiI fluorescence was calculated relative to that in cells pre-treated with PBS.
2.6 Analysis of cell viability after the treatment of HCAECs with pharmacologic
inhibitors
HCAECs (5 × 104 cells/well) were dispensed into 24-well plates and incubated with L5
(50 mg/mL) or PBS for 24 h in the presence or absence of aspirin (0.2 mM), Gi
protein inhibitor pertussis toxin PTX (100 ng/mL), methylation inhibitor
5-aza-deoxycytidine
(5-aza-dC;
0.4
mg/mL),
or
Akt
inhibitor
1L6-hydroxymethyl-chiro-inositol-2-(R)-2-O-methyl-3-Ooctadecylsn-glycerocarbonate (Calbiochem, San Diego, CA, USA) (1 mg/mL). The index of EC
viability was determined by the colorimetric MTT assay (Sigma-Aldrich). Absorbance
was measured at a wavelength of 540 nm by using a microplate reader (Thermo
Electron Corporation, Waltham, MA, USA). Cell viability was calculated relative to
that in the PBS-treated control.
2.7 Luciferase reporter gene assay
The reporter gene assay was performed by using a dual-luciferase expression system
(Promega). Human FGF2 5′-flanking sequences26 were PCR amplified from human
genomic DNA and inserted into the firefly luciferase reporter vector, pGL3-basic, and
were completely sequenced. FGF2 constructs contained the FGF2 promoter
sequence (2126 to +43 or 2126 to +179) or FGF2 gene sequence with the promoter
deleted (+24 to +179). HCAECs were grown to 80% confluence in plastic 12-well
plates and were transfected with 0.75 mg of pGL3-basic or an equimolar amount of
each pGL3-FGF2 construct along with 0.5 mg of the Renilla luciferase expression
vector, phRL-TK, by using Superfect reagent according to the manufacturer’s
instructions (Qiagen, Valencia, CA, USA). Cells were treated 24 h later with PBS or
STEMI L5 (50 mg/ mL) for 24 h in the presence or absence of 0.2 mM aspirin or 0.2
mM indomethacin. Cell lysates were prepared for luciferase assays by using luciferin
and a luminometer (Packard Harvester; Packard Instrument, Meriden, CT, USA). The
promoter activity of the reporter construct was normalized to the promoter activity
of phRL-TK and expressed as a fold-increase relative to that in cells transfected with
pGL3-basic.
2.8 Bisulfite genomic DNA sequencing
HCAECs (cultured in triplicate) were grown to 80 to 90% confluence and then treated
with STEMI L5 (50 mg/mL) in the presence or absence of aspirin (0.2 mM) for 24 h,
followed by genomic DNA extraction using standard procedures. The EpiTect
bisulfite kit (Qiagen) was used to convert all unmethylated cytosine residues in
genomic DNA (2 mg) to uracil. Bisulfite-modified DNA was amplified with
FGF2-specific primers (see Supplementary material online, Table S1) by using the
following cycling conditions: 15 min at 958C followed by 40 cycles of 10 s at 958C
and 45 s at 558C.23 DNA sequencing was performed for 10 plasmid clones from each
treatment group.
2.9 Statistical analysis
The significance of the difference between mean values was assessed by using a
two-way Student t-test for single comparisons and the Bonferroni test for multiple
comparisons. Probability values ,0.05 were considered significant. Results are
expressed as the mean+standard error of the mean.
3. Results
3.1 Plasma L5 levels are significantly elevated in STEMI patients
The characteristics of STEMI patients and control subjects are shown in Table 1. Total
cholesterol and LDL-cholesterol levels were similar between STEMI and control
groups. However, the percentage of L5 LDL (L5/LDL%) and thus the concentration of
plasma L5 ([L5]) were significantly higher in STEMI patients than in control subjects
(P , 0.001) (Table 1, Figure 1A). Agarose gel electrophoresis confirmed the
electronegativity of STEMI L5 (Figure 1B).
3.2 Aspirin attenuates the effects of L5 on FGF2 protein expression and DNA
synthesis
In the clinical setting, peak plasma concentrations of aspirin only reach _0.15 mM,
even after the oral administration of high-dose aspirin (650 mg).27 However, aspirin
concentrations previously used in several in vitro studies have been in the mM range.
Thus, in our experiments, we examined the effects of low- and high-concentration
aspirin. Low-concentration aspirin (0.2 mM) alone did not affect intracellular FGF2
protein levels, but high-concentration aspirin (1–5 mM) alone reduced intracellular
FGF2 protein levels in a concentrationdependent manner (Figure 2A; see
Supplementary material online, Figure S1). Furthermore, 50 mg/mL STEMI L5 or
oxLDL each decreased intracellular FGF2 protein levels by approximately 80%—an
effect that was alleviated by low-concentration aspirin but not highconcentration
aspirin (Figure 2A and B). Parallel with these findings, low-concentration aspirin (0.2
mM) alone had no effect on DNA synthesis in HCAECs, whereas high-concentration
aspirin (1–5 mM) alone inhibited DNA synthesis in HCAECs in a
concentrationdependent manner (Figure 2C). STEMI L5 (50 mg/mL) decreased DNA
synthesis in HCAECs by about 40%—an effect that was prevented by
low-concentration aspirin but not high-concentration aspirin (Figure 2C).
3.3 Low-concentration aspirin attenuates the internalization of L5 by HCAECs and
L5-induced LOX-1 mRNA expression
To determine whether aspirin affects the internalization of L5 by HCAECs, we used
DiI-labelled L5 to visualize intracellular L5. In HCAECs pre-treated with aspirin (0.2
mM), the internalization of DiI-labelled L5 (50 mg/mL) was significantly blocked (P ,
0.05) (Figure 3A). As expected, pre-treatment with anti-LOX-1, which neutralizes the
LOX-1 receptor that mediates the endocytosis of L5, also attenuated the uptake of L5
in HCAECs, whereas pretreatment with anti-LDLR did not (Figure 3A). To determine
whether aspirin effects LOX-1 mRNA expression, we examined LOX-1 mRNA levels in
HCAECs treated with L1, L5, or L5 + 0.2 mM aspirin. Real-time PCR analysis showed
that L5, but not L1, increased LOX-1 mRNA expression and that aspirin partially
reversed the effects of L5 (see Supplementary material online, Figure S2).
3.4 Akt mediates aspirin’s regulation of L5-induced cytotoxicity
To characterize the pathway through which aspirin regulates L5-induced cytotoxicity,
we examined HCAEC viability in the presence or absence of STEMI L5, aspirin (0.2
mM), or pharmacologic inhibitors by using the MTT assay (Figure 3B; see
Supplementary material online, Figure S3). Relative to PBS, STEMI L5 alone reduced
cell viability by about 40% (treatment 1), which was significantly reversed in cells
incubated with PTX (treatment 3) or 5-aza-dC (treatment 4) (Figure 3B). Aspirin (0.2
mM) also reversed the cytotoxic effect of L5 (treatment 2); however, co-treatment
with
the
Akt
inhibitor
1L6-hydroxymethyl-chiro-inositol-2-(R)-2-Omethyl-3-O-octadecyl-sn-glycerocarbonat
e blocked the effect of aspirin (treatment 8 vs. treatment 2). These results indicated
that DNA methylation contributed to the cytotoxicity of L5, which could be
attenuated by 0.2 mM aspirin. In addition, aspirin’s regulation of L5-induced
cytotoxicity was mediated by Akt (Figure 3B).
3.5 L5 and aspirin regulate the FGF2 promoter
To determine whether L5 and aspirin regulate FGF2 protein expression at the
transcriptional level, we evaluated FGF2 mRNA levels in HCAECs treated with L5 in
the presence or absence of aspirin. Lowconcentration aspirin (0.2 mM) but not
high-concentration aspirin (5 mM) attenuated STEMI L5-induced down-regulation of
FGF2 mRNA levels (see Supplementary material online, Figure S4). Furthermore, we
examined the regulation of FGF2 promoter activity by using a luciferase reporter
gene assay (Figure 4A). In the absence of STEMI L5, constructs containing FGF2
sequences –126/+43 and –126/+179 induced 120-fold more luciferase activity than
did the pGL3 vector alone, confirming the presence of a basal promoter between –
126 and +43 of FGF2. Addition of STEMI L5 (50 mg/mL) reduced the luciferase
activity of constructs –126/+43 and –126/+179 by 30 to 40%, respectively (P , 0.05).
This reduction was reversed in the presence of aspirin (0.2 mM) but not
indomethacin (a cyclooxygenase inhibitor with antiplatelet activity) (Figure 4B). Thus,
in HCAECs, physiologic levels of aspirin suppressed the effects of L5 on FGF2
transcription through a cyclooxygenase-independent mechanism. Furthermore, the
attenuation of L5-induced FGF2 promoter suppression by aspirin was partially
prevented by Akt inhibitor (see Supplementary material online, Figure S5),
suggesting the involvement of Akt in the
regulation of FGF2 by aspirin and L5.
3.6 L5 and aspirin regulate the FGF2 promoter via methylation of a CpG island
Genomic DNA sequence analysis was used to identify an 1877-bp CpG island in the
human FGF2 gene that starts at –532 in the 5′-flanking region of FGF2 and extends
through exon 1 into the first intron. This portion of the human FGF2 gene contains
the L5-responsive promoter that we show in Figure 4B can be regulated by aspirin
(Figure 5A). Because CpG methylation is a key factor in FGF2 gene expression,23 we
investigated whether L5 and aspirin regulate CpG methylation of the FGF2 promoter.
The methylation status of 20 CpG dinucleotides in the FGF2 promoter was
characterized by using bisulfite genomic DNA sequencing in HCAECs (Figure 5B).
None of the 20 cytosine residues was methylated in the PBS-treated control cells. In
contrast, the treatment of cells with STEMI L5 (50 mg/mL) resulted in the
methylation of all 20 cytosine residues. When 0.2 mM aspirin was added to
L5-treated HCAECs, the methylation of these cytosine residues was markedly
reduced: only 8 of the 20 remained methylated. In addition, we examined the mRNA
levels of DNA methyltransferase (DNMT)1, DNMT3A, and DNMT3B by real-time PCR
and observed a modest L5-induced increase in the levels of DNMT1 and DNMT3B
mRNA—an effect that could be attenuated by aspirin (see Supplementary material
online, Figure S6). These data indicated that L5 represses FGF2 transcription in ECs
by promoting the methylation of CpG dinucleotides in the FGF2 promoter and that
this effect is significantly reduced by the addition of aspirin.
4. Discussion
We have shown for the first time that L5 is significantly increased in the plasma of
STEMI patients when compared with that of control subjects. In our STEMI patients,
the mean plasma L5 level was increased to _12% of total LDL. The mean plasma [L5]
in these patients was approximately 150 mg/mL, which far exceeds the toxic
threshold of L5 (25–50 mg/mL) determined in vitro,8,12,13,16 helping to explain
why severe coronary endothelial dysfunction occurs in STEMI.28 We have found that
patients with non-ST elevation myocardial infarction (NSTEMI) have lower and more
variable L5 levels than patients with STEMI, ranging between 0.5 and 5% of total LDL
(unpublished data). In addition, we found that L5 isolated from STEMI patients can
impair coronary endothelial function by inducing methylation of CpG sites in the
FGF2 promoter. Importantly, we showed that low-concentration aspirin can
suppress the L5-induced methylation of FGF2. Equally as important,
high-concentration aspirin not only lost this protective capability but accentuated
the harmful effects of L5. Our findings are summarized in Figure 6. An indicator of EC
survival,29 FGF2, is a potent angiogenic factor involved in all aspects of angiogenesis
(e.g. EC proliferation and migration and vascular differentiation). Previously, we
showed that both oxLDL and electronegative L5 LDL down-regulated endothelial
FGF2 by inhibiting Akt phosphorylation.10 In this study, the ability of an Akt inhibitor
to attenuate the effects of aspirin on L5-reduced FGF2 expression further supports
the important role of Akt in the L5 signalling pathway. Furthermore, involvement of
the Akt and Gi signaling pathways in the regulation of the FGF2 promoter by L5 and
aspirin also implies a complex interplay between cellular survival and different
signalling pathways in the response to atherogenic L5 and aspirin. Our results
showed that aspirin preserves EC function by suppressing FGF2 promoter
methylation by L5. As we have shown in the current study, one mechanism by which
aspirin may counteract the effects of L5 is by preventing the uptake of L5 into ECs.
Furthermore, we showed that the CpG-rich promoter of FGF2 is heavily methylated
in the presence of STEMI L5 and that this effect is suppressed by the addition of
low-concentration aspirin, suggesting that aspirin may regulate FGF2 through an
epigenetic mechanism of action. Promoter DNA methylation by L5 has not been
previously reported and may be not limited to the promoter of FGF2. We have
similarly studied promoter methylation of the apoptosis-related LOX-1 and Bcl-2
genes and found that L5-induced cytosine methylation occurred at cytosine residues
in the LOX-1 promoter but not in the Bcl-2 promoter (unpublished data). The
5′-flanking region of the FGF2 promoter is responsible for many activities of this gene,
including its basal transcription.26 The FGF2 promoter is located within a CpG island,
which is defined as a region of DNA larger than 500 bp that has a moving average
%(G+C) .55 and an observed/expected CpG dinucleotide ratio .0.65.30 The human
FGF2 gene contains multiple GC boxes (GGGCGG or CCGCCC) and a basal promoter
between 2126 and +24, which was vulnerable to modulation by STEMI L5 and aspirin
in opposite directions in our transient transfection system. Recently, chronic aspirin
use (,300 mg/day) has been associated with reduced CpG methylation of the
promoter of E-cadherin (an adhesion molecule involved in tumour invasion and
metastasis), providing support that aspirin is capable of epigenetic regulation.31
The protective effect of low-dose aspirin (75–325 mg/day) on the cardiovascular
system has been largely attributed to aspirin’s anti-thrombotic activity as an
irreversible inhibitor of cyclooxygenase-1 (COX-1) in platelets.2,32 However, it has
been shown that aspirin has pleiotropic effects on vascular ECs that are induced
through a cyclooxygenase-independent mechanism,3,4 which may explain the
complex anti-atherogenic and anti-inflammatory effects of aspirin. Thus, it is
plausible that the beneficial effects of aspirin that have been seen in primary
prevention trials of acute coronary events were not caused by aspirin’s conventional
inhibitory effects on platelet aggregation, leucocyte adhesion, or smooth muscle cell
proliferation, but by a novel vasoprotective action, such as improving EC survival.
However, the interplay between low-concentration aspirin and coronary
atherosclerosis is complex. In some clinical trials, the ‘protective’ plasma levels of
0.1–0.2 mM aspirin, which we found to be beneficial in our in vitro experiments,
failed to successfully prevent restenosis after vascular angioplasty.33,34 The
discrepancy between the prosurvival effect of 0.2 mM aspirin on HCAECs observed in
this study and the lack of an anti-thrombotic effect of similar therapeutic
concentrations of aspirin observed in clinical trials underlies the need for complex in
vitro models that mimic the clinical situation more closely. In conclusion, we have
characterized a novel mechanism of aspirin in protecting the coronary endothelium
against the effects of L5. Our findings suggest that aspirin’s regulation of FGF2 may
be as important as its anti-platelet activity in preventing MI. Moreover, our evidence
that a therapeutic concentration but not high concentrations of aspirin protect ECs
from L5-induced cell death provides important groundwork for developing a
targeted therapeutic approach for the prevention of plaque instability.
Supplementary material
Supplementary material is available at Cardiovascular Research online.
Acknowledgements
The authors thank Nicole Stancel, PhD, ELS, of the Texas Heart Institute at St Luke’s
Episcopal Hospital, for editorial assistance in the preparation of this manuscript.
Conflict of interest: none declared.
Funding
This work was supported by grants NSC 91-2320-B-002-185, 93-2314-B-002-125,
94-2320-B-002-121,
95-2320-B-002-116,
98-2628-B-002-088,
97-2320-B-002-057-MY3 to (P.-Y.C., Y.-T.L.,J.L.) and NSC 100-2314-B-039-040-MY3
(C.-H.C.) from the National Science Council, Taipei, Taiwan; grants NTUH 92A14,
93A02, 95S342 from the National Taiwan University Hospital, Taipei, Taiwan to
P.-Y.C.; Taiwan Department of Health Clinical Trial and Research Center of Excellence,
DOH102-TD-B-111-004 to C.-H.C.; and research grant 1-04-RA-13 from the American
Diabetes Association to C.-H.C. The authors have no relationships with industry to
declare.
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