Anti-inflammatory and Antioxidant Properties of HDLs Are Impaired in Type 2 Diabetes
Alan M. Fogelman
Srinivasa T. Reddy
Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, University of California Los Angeles
Los Angeles, California
Department of Medicine, David Geffen School of Medicine, University of California Los Angeles
Los Angeles, California
; and the
Department of Internal Medicine, University of Pisa
OBJECTIVEIn mice, 4F, an apolipoprotein A-I mimetic peptide that restores HDL function, prevents diabetes-induced atherosclerosis. We sought to determine whether HDL function is impaired in type 2 diabetic (T2D) patients and whether 4F treatment improves HDL function in T2D patient plasma in vitro. RESEARCH DESIGN AND METHODSHDL anti-inflammatory function was determined in 93 T2D patients and 31 control subjects as the ability of test HDLs to inhibit LDL-induced monocyte chemotactic activity in human aortic endothelial cell monolayers. The HDL antioxidant properties were measured using a cell-free assay that uses dichlorofluorescein diacetate. Oxidized fatty acids in HDLs were measured by liquid chromatography-tandem mass spectrometry. In subgroups of patients and control subjects, the HDL inflammatory index was repeated after incubation with L-4F. diabetes.diabetesjournals.org
RESULTSThe HDL inflammatory index was 1.42 6 0.29 in T2D
patients and 0.70 6 0.19 in control subjects (P , 0.001). The
cellfree assay was impaired in T2D patients compared with control
subjects (2.03 6 1.35 vs. 1.60 6 0.80, P , 0.05), and also HDL
intrinsic oxidation (cell-free assay without LDL) was higher in
T2D patients (1,708 6 739 vs. 1,233 6 601 relative fluorescence
units, P , 0.001). All measured oxidized fatty acids were
significantly higher in the HDLs of T2D patients. There was a significant
correlation between the cell-free assay values and the content of
oxidized fatty acids in HDL fractions. L-4F treatment restored the
HDL inflammatory index in diabetic plasma samples (from 1.26 6
0.17 to 0.71 6 0.11, P , 0.001) and marginally affected it in
healthy subjects (from 0.81 6 0.16 to 0.66 6 0.10, P , 0.05).
CONCLUSIONSIn patients with T2D, the content of oxidized
fatty acids is increased and the anti-inflammatory and antioxidant
activities of HDLs are impaired. Diabetes 60:26172623, 2011
Tof vascular events compared with nondiabetic
ype 2 diabetic (T2D) patients remain at higher risk
subjects, despite achieving recommended targets
of serum cholesterol, blood pressure, and
glycemia; therefore, other factors must be involved in this risk
inherent in the diabetic condition (1). Recent evidence
suggests that circulating lipoproteins might significantly differ
in their biological activities in relation to vascular function
(24). We hypothesized that qualitative, in addition to
quantitative, differences in lipoproteins might be one of the factors
responsible for the unexplained residual risk in diabetic
patients, especially because lipoproteins are associated with
inflammation and oxidative stress, which are considered to
be important steps in atherosclerosis development.
VLDL and LDL particles isolated from patients with T2D
or metabolic syndrome have an increased susceptibility to
lipolysis, and this results in a higher concentration of
nonesterified fatty acids and increased content of
lysophosphatidylcholine in lipoproteins (5); both of these bioactive
lipids may contribute to the proinflammatory state in these
individuals. Oxidized LDL induces the release of
endotheliumderived inflammatory mediators and the expression of
adhesion molecules (3,6). HDL has been shown to protect
LDL from oxidation (79), and in a coculture of human
endothelial cells and human smooth muscle cells, HDL is
able to inhibit the LDL-induced production of the potent
monocyte chemoattractant, monocyte chemotactic protein-1,
and the migration of monocytes (10). Van Lenten et al. (11)
demonstrated that HDL could act as an anti-inflammatory
molecule or a proinflammatory molecule, depending on
the context and environment. These authors have shown
that during an acute-phase response both in animals
(rabbits) and in humans, HDL is converted from an
antiinflammatory to a proinflammatory (11). In subjects with
T2D, increased levels of markers of chronic low-grade
inflammation, such as C-reactive protein (CRP) and serum
amyloid A (SAA), might be implicated in driving qualitative
changes in HDLs (12). This modification in HDL
composition and function would not only lower the capacity of
HDLs to protect LDLs against oxidative modification but
also their ability to protect the vessel from the negative
effect of oxidized LDLs. We recently published that an
apolipoprotein (apo) A-I mimetic peptide (D-4F) was able
to bind oxidized lipids with much higher affinity than
apoA-I (13), reduced atherosclerosis development, and
prevented diabetes-induced oxidized lipid accumulation in
a mouse model of diabetes (14).
In the present work, we demonstrate that HDL
antioxidant and anti-inflammatory properties are impaired in T2D
patients compared with healthy control subjects. We
additionally show that the apoA-I-mimetic peptide L-4F can
alter the quality of HDLs in diabetic patients ex vivo.
RESEARCH DESIGN AND METHODS
Ninety-three patients with T2D attending the outpatient clinic of the
Department of Internal Medicine (University of Pisa, Pisa, Italy) were recruited
within a 12-month period. Exclusion criteria were as follows: any acute and
chronic inflammatory disease, any recent (6 months) cardiovascular event, any
previous diagnosis of cancer, moderate to severe chronic kidney or liver
disease, and regular or frequent use of anti-inflammatory drugs or antioxidants.
Thirty-one healthy control subjects (age- and sex-matched) were recruited from
the relatives of the patients and from the personnel of the Department of
Internal Medicine. After participants gave their written informed consent, a visit
was scheduled within 7 days in fasting conditions for blood sampling (~80 mL),
which was followed by a visit and an interview done by the same investigator to
collect cardiovascular risk and health information.
Biochemical measurements. Blood was collected in specific tubes for routine
biochemistry (HbA1c, lipids, liver function tests, creatinine, and CRP) and in
four 10-mL EDTA tubes (40 mL) that were immediately centrifuged to collect
the plasma that was stored in 1-mL aliquots at 280C for the subsequent
analysis. All the assays were performed in a double-blinded manner.
SAA. SAA levels were determined using a Human SAA ELISA kit (no. KHA0012;
Invitrogen, Carlsbad, CA).
HDL inflammatory index. HDL ability to interfere with LDL-induced
monocyte migration was measured using the monocyte chemotactic activity (MCA)
assay (10). In brief, human aortic endothelial cell cultures (HAECs) were
obtained from the trimmings of a donor aorta during a heart transplant according
to University of California Los Angeles Institutional Board guidelines. Standard
LDL (sLDL) was isolated from the plasma of normal blood donors by
densitygradient ultracentrifugation, and human peripheral blood monocytes were
obtained from normal healthy donors. The HAECs were grown as monolayers in
culture and were treated with sLDLs (100 mg/mL LDL cholesterol) in the
absence or presence of test HDLs (50 mg/mL) overnight. Solutions of LDLs or
HDLs, fractionated by fast-performance liquid chromatography (FPLC), were
diluted with culture medium 199 containing 10% lipoprotein-deficient serum
obtained from healthy volunteers and added to culture wells. After the
incubation, the supernatants were collected from the cultures, diluted 20-fold, and
assayed for MCA, as described previously (15). In brief, the supernatants were
added to a standard NeuroProbe chamber (NeuroProbe, Cabin John, MD), with
isolated human peripheral blood monocytes added to the top. The chamber was
incubated for 60 min at 37C. After the incubation, the chamber was
disassembled, and the nonmigrated monocytes were removed. The membrane was
then air-dried and fixed with 1% glutaraldehyde and stained with 0.1% crystal
violet dye. The number of migrated monocytes was determined microscopically
in six standardized high-power fields counted in triplicate wells. The values
obtained with test HDLs were divided by the value obtained with LDLs alone.
The addition of anti-inflammatory HDLs reduces LDL-induced MCA, and this
results in a value ,1.0. Proinflammatory HDL conversely results in a value .1.0.
LDL inflammatory index. For determination of the LDL inflammatory index,
the test LDL was added to the cells without added HDLs, and the resulting MCA
was divided by the MCA obtained after addition of sLDLs without added HDLs,
as previously described (15).
Plasma inflammatory index. For determination of the plasma index, whole
plasma from patients and healthy control subjects (diluted 1:2,000 using culture
medium) was incubated with HAECs, and MCA assay was performed. The index,
similar to the HDL inflammatory index, was calculated normalizing the migration
values obtained with the test plasma by the value of a standard reference plasma
sample obtained from healthy donors.
HDL antioxidant properties were measured by 1) the cell-free assay
described previously (16) as the ratio of HDL + sLDL fluorescence to sLDL alone
and 2) HDL intrinsic oxidation, which is measured by performing the cell-free
assay on HDLs alone (i.e., without sLDLs). HDLs used for these experiments
were isolated by the dextran sulfate method. In brief, 50 mL HDL Magnetic
Bead Reagent (Polymedco, Cortland Manor, NY) were mixed with 250 mL of
the subjects plasma and first incubated for 5 min at room temperature then for
an additional 5 min on a magnetic particle concentrator. HDL cholesterol in
the supernatant was quantified using a standard assay (Thermo DMA, San
Jose, CA) (11).
The cell-free assay was performed, as described previously (16), with slight
modification. In brief, 25 mL of standard LDL solution containing 2.5 mg LDL
cholesterol and HDLs from each patient at two different concentrations (2.5
and 5 mg HDL cholesterol in 125 mL PBS) were incubated in a 96-well plate for
30 min at 37C. Then, 25 mL 2,7-dichlorfluorescein (DCFH) solution were
added to each well, and after 60 min of incubation at 37C, fluorescence
intensity was measured. Values for the fluorescence intensity induced by test
HDL + sLDL were divided by the values obtained with sLDLs alone to obtain
an index value. Index values $1.0 indicate dysfunctional HDLs (pro-oxidant
HDL), while values ,1.0 indicates normal, antioxidant HDLs (16).
HDL intrinsic oxidation was measured by the fluorescence intensity resulting
from the interaction DCFH with HDLs alone. HDLs from each patient at two
different concentrations (2.5 and 5 mg HDL cholesterol in 150 mL PBS) were
incubated with 25 mL DCFH solution (0.2 mg/mL) for 60 min of incubation at
37C. The results are expressed as relative fluorescence units (rfu).
Determination of free fatty acids in HDL-containing fractions. Liquid
chromatographytandem mass spectrometry (LC/MS/MS) was performed
using a quadruple mass spectrometer (4000 QTRAP; Applied Biosystems, Foster
City, CA) equipped with an electrospray ionization source. Chromatography
was performed using a Luna C-18 column (3-mm particle, 150 3 3.0 mm;
Phenomenex, Inc., Torrance, CA) with a security-guard cartridge (C-18;
Phenomenex, Inc.) at 40C. Detection was accomplished by using the
multiplereactionmonitoring mode with negative ion detection.
Randomly chosen sets of samples from patients and control subjects were
both thawed the day of the experiment and processed under identical conditions.
HDLs (50 mg cholesterol) in 1.8 mL FPLC buffer were spiked with 1.8 mL of 20
mmol/L butylated hydroxytoluene (BHT) in ethanol and 100 mL internal standard
mixture [15(S)-HETE-d8, 12(S)-HETE-d8, 5(S)-HETE-d8, and 13(S)-HODE-d4, 10
ng/mL each] in methanol. The sample was loaded onto a preconditioned Oasis
HLB solid-phase extraction cartridge (1 mL, 10 mg) on a vacuum manifold
(Waters Corporation, Milford, MA). The solid-phase extraction cartridge was
equilibrated with 1 mL methanol followed by 1 mL water before the sample load.
After the sample loading, the cartridge was washed with 1 mL 5% methanol in
water, and free fatty acids were subsequently eluted with 1 mL methanol. The
eluate was evaporated under argon and reconstituted with 60 mL methanol,
vortexed, and transferred to an autosampler vial for LC/MS/MS analysis. LC/MS/
MS analysis was performed as described previously (17). The transitions
monitored were mass-to-charge ratio (m/z): 319.1179.0 for 12-HETE; 319.1219.0
for 15-HETE; 295.0194.8 for 13-HODE; 319.1115.0 for 5-HETE; 295.0171.0
for 9-HODE; 327.1226.1 for 15(S)-HETE-d8; 327.1184.0 for 12(S)-HETE-d8;
299.0 197.9 for 13(S)-HODE-d4; and 327.1115.9 for 5(S)-HETE-d8. The
following chemicals were used: (6)12-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic
acid (12-HETE); (6) 15-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic acid (15-HETE);
(6)13-hydroxy-9Z,11E-octadecadienoic acid (13-HODE);
(6)9-hydroxy-10E,12Zoctadecadienoic acid (9-HODE); (6)5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic
acid (5-HETE); 12(S)-hydroxy-5Z,8Z,10E,14Z-eicosatetraenoic-5,6,8,9,11,12,14,
15-d8 acid (12(S)-HETE-d8);
15(S)-hydroxy-5Z,8Z,11Z,13E-eicosatetraenoic5,6,8,9,11,12,14,15-d8 acid (15(S)-HETE-d8); 5(S)-hydroxy-6E,8Z,11Z,
14Z -eicosatetraenoic-5,6,8,9,11,12,14,15-d8 acid (5(S)-HETE-d8); and
13(S)hydroxy-9Z,11E-octadecadienoic-9,10,12,13-d4 acid (13(S)-HODE-d4)
(purchased from Cayman Chemicals, Ann Arbor, MI). High-performance liquid
chromatographygrade methanol was obtained from Sigma-Aldrich (St. Louis,
MO). High-performance liquid chromatographygrade acetonitrile was obtained
from Fisher Scientific (Pittsburgh, PA). Oasis HLB was purchased from Waters
Ex vivo treatment with L-4F. Ten plasma samples from patients and 10
plasma samples from healthy control subjects were randomly chosen and
treated with buffer or with L-4F at 0.5 mg peptide per milliliter of plasma. For
determination of the plasma inflammatory index, plasma aliquots were
incubated with L-4F for 15 min at 37C with gentle rotation and then diluted with
culture medium. For determination of the HDL inflammatory index and LDL
inflammatory index, plasma aliquots were incubated for 15 min at 37C with
gentle rotation and then fractionated by FPLC to obtain LDL and HDL
fractions. The diluted plasma, HDLs, or LDLs then were incubated with HAEC
monolayers, and the HDL inflammatory index and LDL inflammatory index
were determined as described above.
Data analysis. Data were expressed as means 6 SD, unless otherwise
indicated. Differences between values for patients and healthy control subjects
were determined by ANOVA with JMP software (JMP version 7.0).
The study population characteristics are described in
Table 1. Patients were obese (BMI 34 6 8 kg/m2), 80% had
a diagnosis of arterial hypertension, and 13% had a
previous major cardiac event. Biochemical characteristics are
shown in Table 2. Patients were in suboptimal glycemic
control (HbA1c 8 6 2%), and lipid profiles were abnormal
despite 58% of them being treated with either statins or
fibrates. Diabetic patients also had elevated CRP levels.
Healthy volunteers had a very low cardiovascular risk,
with neither diabetes nor high blood pressure, and normal
lipid profile. None of the healthy volunteers were on
medications or vitamin supplementations.
Data are means 6 SD, unless otherwise indicated. *P , 0.05 patients
vs. control subjects.
Data are means 6 SD. ND, not determined. eGFR, estimated
ular filtration rate using the Modification of Diet Renal Disease
(MDRD) formula. AST, aspartate aminotransferase. ALT, alanine
aminotransferase. GGT, g-glutamyl transpeptidase.
SAA. Plasma SAA was measured in a subgroup of 26
diabetic patients and 24 healthy control subjects. SAA plasma
concentrations were increased in diabetic patients
compared with healthy control subjects (48.2 6 35.1 mg/mL vs.
22.7 6 1.5 mg/mL, P , 0.005) (Fig. 1).
HDL inflammatory index. Compared with the HDLs of
healthy volunteers, HDLs from diabetic patients were less
able to inhibit the migration of monocytes induced by
LDLs (Fig. 2A). The mean HDL inflammatory index value
in patients with diabetes was significantly .1.0 (1.42 6
0.29), indicating not only the loss of anti-inflammatory
property but also an increase in proinflammatory HDLs.
FIG. 1. Increased plasma SAA levels in diabetic patients compared with
control subjects. The SAA plasma concentrations were measured
(as described in RESEARCH DESIGN AND METHODS) in 26 diabetic patients
and 24 healthy control subjects. *P < 0.05 patient vs. control subject.
LDL inflammatory index. LDLs from patients with T2D
induced a greater MCA compared with healthy control
subjects. The LDL inflammatory index was 1.24 6 0.15 in
patients with diabetes and 1.07 6 0.09 in control subjects
(P , 0.001) (Fig. 2B).
Plasma inflammatory index. The plasma of patients with
diabetes, as a whole, exerted a proinflammatory activity
(Fig. 2C); the plasma inflammatory index was not only
significantly higher than in healthy volunteers (1.30 6 0.17 vs.
1.17 6 0.13, P , 0.001) but also was significantly .1,
implying a nonneutral activity.
HDL antioxidant properties. The HDL antioxidant
properties were examined in 73 diabetic and 31 control subjects.
The index obtained from the cell-free assay (as the ratio
of sLDL + HDL fluorescence to sLDLs alone) was
significantly higher in the diabetic patients compared with healthy
volunteers, both when HDL cholesterol was added at low
(2.5 mg/125 mL) and high (5.0 mg/125 mL) concentrations
(2.03 6 1.35 vs. 1.60 6 0.80, P , 0.05, and 1.50 6 0.99 vs.
1.11 6 0.44, P , 0.01, respectively). Moreover, HDLs from
diabetic subjects incubated with DCFH directly (without
LDL) showed increased fluorescence values compared with
healthy volunteers (1,708 6 739 rfu vs. 1,233 6 601 rfu, P ,
0.001; 2.5 mg HDL cholesterol was used for this assay),
indicating an increased intrinsic HDL oxidation.
Oxidized fatty acids in HDL-containing fractions. As
shown in Table 3, fatty acids resulting from oxidation of
arachidonic acid (12-HETE, 15-HETE, and 5-HETE) and
linoleic acid (9-HODE and 13-HODE) were significantly
elevated in HDLs isolated from 11 diabetic patients
compared with HDLs from 8 randomly selected control
subjects. Despite the small numbers in the subset of subjects
for which the content of oxidized fatty acids was
determined, there was a significant correlation between the
cell-free assay values and the content of oxidized fatty
acids in HDL fractions (12-HETE [r2 = 0.28, P = 0.01];
5-HETE [r2 = 0.56, P = 0.0002]; 15-HETE [r2 = 0.45, P =
0.002]; 9-HODE [r2 = 0.66, P , 0.0001]; and 13-HODE
[r2 = 0.70, P , 0.0001]).
Ex vivo treatment with L-4F. Plasma samples from 10
diabetic subjects and 10 control subjects were treated with
L-4F ex vivo. Incubation with L-4F induced a significant
improvement in the HDL inflammatory index in diabetic
patients (buffer: 1.26 6 0.17; L-4F: 0.71 6 0.11; P , 0.001)
and also in healthy volunteers (buffer: 0.81 6 0.16; L-4F:
0.66 6 0.10; P = 0.02) (Fig. 3A). In addition, L-4F treatment
reduced the LDL inflammatory index in patients (buffer:
1.29 6 0.17; L-4F: 0.99 6 0.34; P = 0.02) and in healthy
volunteers (buffer: 1.10 6 0.04; L-4F: 1.04 6 0.06; P = 0.01)
(Fig. 3B). L-4F also was able to reduce the plasma
inflammatory index in both diabetic subjects (buffer: 1.31 6
0.20; L-4F: 0.95 6 0.18; P = 0.0006) and control subjects
(buffer: 1.14 6 0.11; L-4F: 0.96 6 0.16; P = 0.009) (Fig. 3C).
The main finding of this study is that patients with T2D have
a chronic inflammatory condition that is characterized not
only by increased levels of acute-phase proteins (CRP and
SAA) but also by enhanced LDL proinflammatory properties
linked with the loss of the anti-inflammatory and
antioxidant effect of their HDLs. Moreover, in our data, a
statistically significant correlation was found between HDL
inflammatory index values and SAA plasma concentrations
(r2 = 0.09, P = 0.02). It has been shown that there is a
strong relationship between LDL and HDL inflammatory
properties and atherosclerotic lesions in cholesterol-fed
FIG. 2. The HDL inflammatory index (A), LDL inflammatory index (B), and plasma inflammatory index (C) were significantly higher in diabetic
patients compared with control subjects. The HDL, LDL, and plasma inflammatory indices were determined using a monocyte chemotactic assay in
the whole population of the study (93 diabetic subjects and 31 healthy control subjects) (as described in RESEARCH DESIGN AND METHODS). *P < 0.05
patients vs. control subjects.
rabbits (15). In humans, the HDL inflammatory index also
is significantly correlated with intima media thickening
and atherosclerotic plaque size in patients with systemic
lupus erythematous (18).
The cell-free assay data suggest that oxidative stress
is important in mediating these changes in diabetic
lipoproteins. The presence of oxidized lipids in HDLs has been
proposed to be responsible for functional changes in HDLs
(10). Excess generation of reactive oxygen species by
hyperglycemia (19) might be involved in the enhanced
production of oxidized lipids from arachidonic and linoleic
acid in diabetic lipoproteins. The results in Table 3 provide
some of the first evidence of increased oxidized fatty acids
in human diabetic HDLs. Although the size of the subset of
subjects analyzed for oxidized fatty acids was small, there
was a statistically significant positive correlation between
the HDL antioxidant index measured by the cell-free assay
and the content of oxidized fatty acids in HDL fractions. Of
12-HETE (ng/50 mg HDL)
5-HETE (ng/50 mg HDL)
15-HETE (ng/50 mg HDL)
9-HODE (ng/50 mg HDL)
13-HODE (ng/50 mg HDL)
Data are means 6 SD. *P , 0.01 patients vs. control subjects.
interest, neither hyperglycemia nor glycated hemoglobin
nor other classical risk factors were correlated with HDL
dysfunction in this study (data not shown).
Results from the ex vivo treatment with L-4F are
consistent with the hypothesis that oxidized lipids in HDLs and
LDLs are responsible for the results reported here, because
the effect of this apoA-I mimetic is related to its ability to
avidly bind oxidized lipids (13).
Low-grade inflammation, as indicated by elevated plasma
SAA and CRP, is likely to enhance lipid oxidation in patients
with diabetes. Acute-phase proteins like SAA and the
haptoglobin-hemoglobin complex have been found on HDL,
and their presence has been shown to favor lipid oxidation
and HDL dysfunction (20). The clinical relevance of these
mechanisms is indirectly supported by the recent analysis
of the Heart Outcomes Prevention Evaluation (HOPE) study,
showing that, in T2D patients, treatment with vitamin E
decreased cardiovascular death by 50% in those with the
haptoglobin genotype 2-2 (20), whereas no effect was
observed in haptoglobin genotype 1-1.
A limitation of this study is that we only compared
diabetic patients to healthy volunteers. Thus, we cannot
determine whether the lipoprotein abnormalities identified
are simply markers of the disease or are causal in their
clinical consequences. The number of diabetic subjects
studied did not allow us to compare the HDL inflammatory
index in diabetic subjects matched for arterial
hypertension, BMI, or antecedent cardiovascular events. Another
limitation of this study was that the antioxidant, BHT, was
added after the collection of plasma and before analysis.
Thus, we cannot exclude the possibility that there was
oxidation during storage. Unterwurzacher et al. (21) added
BHT to plasma and determined the content of free
oxidized fatty acids in patients with diabetes per milliliter of
plasma and found that the plasma content of 9-HODE
and 15-HETE were elevated in diabetic plasma. Our data
were determined as nanograms per 50 mg/HDL cholesterol,
making direct comparisons of exact values between the
two studies difficult. However, the patient and control
samples in our study were treated exactly the same and
analyzed on the same day so that the relative differences
would be valid. Moreover, our finding that diabetic
subjects had higher values is consistent with the findings of
Unterwurzacher et al. (21).
HDL was isolated by FPLC in our studies, and, thus, we
cannot determine how much of the oxidized fatty acids
were associated with albumin, which is known to be part
of the HDL proteome and which was not different in HDL
from coronary heart disease (CHD) patients and control
subjects in the studies of Vaisar et al. (22). Because the
patient and control samples were treated identically in our
studies, the differences remain significant, but the exact
distribution of the oxidized fatty acids within the HDL
FIG. 3. Ex vivo treatment with L-4F rescued HDL function and normalized
the LDL inflammatory index and plasma inflammatory index in diabetic
patient samples and control subjects. A: The effect of L-4F treatment
on the HDL inflammatory index. B: LDL inflammatory index. C: Plasma
inflammatory index. n = 10 per group (diabetic patients and healthy
proteome will need to be determined in future studies. In
addition, these studies did not differentiate between the
HDLs of different sizes. Future studies will need to
determine whether HDL size correlates with the defects in
HDL function described here.
Bloedon et al. (23) reported that oral doses of the 4F
peptide of 4.3 and 7.14 mg/kg significantly improved the
HDL inflammatory index in patients with CHD or
equivalents such as diabetes compared with placebo. However,
Bloedon et al. (23) did not see any significant improvement
in the HDL inflammatory index of these patients compared
with placebo when doses of 0.43 or 1.43 mg/kg were
administered. Watson et al. (24) targeted preset plasma
peptide levels and administered 0.43 mg/kg of the 4F peptide
intravenously or subcutaneously. Although Watson et al.
(24) achieved these preset plasma peptide levels, which
greatly exceeded those achieved at the high doses
administered by Bloedon et al. (23), there was no significant
improvement in the HDL inflammatory index compared
with placebo. Subsequently, Navab et al. (25) demonstrated
that in vivo efficacy of the 4F peptide is determined by the
dose administered and not by the plasma level achieved.
Moreover, Navab et al. (25) found evidence that in vivo the
intestine may be an important site of action for the peptide
whether it is administered orally or by injection.
The HDL inflammatory index has been shown to be
significantly increased (i.e., .1.0) in patients with CHD or
equivalents, compared with healthy control subjects in
three separate studies (23,24,26). In previous studies, we
have shown that the HDL inflammatory index is inversely
correlated with the ability of HDL to mediate cellular
cholesterol efflux (27). Vaisar et al. (22) demonstrated that the
HDL proteome is abnormal in such patients and is
sistent with an inflammatory phenotype. Shao and Heinecke
(28) have demonstrated that HDL oxidation by the
myeloperoxidase system impairs the ability of apoA-I to mediate
sterol efflux by the ABCA1 pathway, and Undurti et al. (29)
reported that modification of HDL by myeloperoxidase
generates a proinflammatory particle. Thus, there is
increasing evidence that the HDL proteome and HDL function
are impaired in CHD.
In conclusion, the finding in our studies that the HDL
inflammatory index in patients with diabetes is, on average,
.1 indicates that, in these patients, HDL dysfunction is such
that HDLs not only have lost their anti-inflammatory activity
but they exert a proinflammatory activity. Whether these
changes also have an impact on other functions of HDL,
such as reverse cholesterol transport, endothelial function,
and platelet aggregation in patients with diabetes, remains
to be determined.
Because monocyte migration is only one of the many
steps involved in the complex process of atherosclerosis,
the effect of an intervention aimed at restoring HDL
antiinflammatory function in patients with diabetes is difficult
to predict. However, because monocyte migration is one of
the earliest steps in the generation of atherosclerotic lesions
and because such interventions would likely also restore
HDL antioxidant activity, such a therapeutic strategy,
particularly if applied early in the natural course of the disease,
might well be beneficial.
This work was supported in part by the U.S. Public Health
Service Grants HL-30568 and HL-082823; the Laubisch,
Castera, and M.K. Grey Funds at the University of California
Los Angeles (UCLA); a network grant from Fondation
Leducq; and an Internal Medicine School of Specialty
Fellowship (to C.M.) from the University of Pisa.
M.N., A.M.F., and S.T.R. are principal investigators at
Bruin Pharma, and A.M.F. is an officer at Bruin Pharma.
No other potential conflicts of interest relevant to this
article were reported.
C.M. recruited patients, researched data, contributed to
the discussion, and wrote and reviewed the manuscript.
A.N. researched data, contributed to the discussion, and
reviewed the manuscript. B.B. recruited patients. S.I.
researched data. M.N. researched data and reviewed the
manuscript. A.M.F. and E.F. reviewed the manuscript. S.T.R.
contributed to the discussion and reviewed the manuscript.
Parts of this study were presented in oral form at the
71st Scientific Sessions of the American Diabetes Associ
ation, San Diego, California, 2428 June 2011.
The authors thank Yen Yin Lee, Department of
Medicine (UCLA), for expert technical assistance and Marion
Benquet, Department of Medicine (UCLA), for the help in