# CD36 Is Significantly Correlated with Adipophilin in Human Carotid Lesions and Inversely Correlated with Plasma ApoAI

Authors: Sophie Collot-Teixeira; Calypso Barbatis; Florence Bultelle; Michael Koutouzis; Gerard Pasterkamp; Paul Fraser; Zenon S. Kyriakides; Robin N. Poston; Angelique Ristagno; Lilian McGregor; Chantal M. Boulanger; Guy Leseche; John L. McGregor
Journal: Journal of Biomedicine and Biotechnology
 (2008)

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## Abstract

OxLDL uptake and cholesterol efflux inhibition in macrophages play a key role in atherosclerotic plaque formation, rupture, and thrombotic ischemia. This study investigates genes implicated in OxLDL uptake (CD36, SRA), cholesterol efflux inhibition (adipophilin, ADFP), and inflammatory recruitments of leukocytes (IL-8) in plaque lesion areas (PLAs) compared to nonplaque lesion areas (NPLAs) in human carotid endarterectomy specimens. Gene and protein expressions were assayed using quantitative PCR and quantitative immunohistochemistry. Pearson tests were used to investigate potential correlation between (a) different gene expressions and (b) gene expression and patient's plasma constituents. CD36, SRA, ADFP, and IL-8 were shown to be significantly more expressed in PLA compared to NPLA. In PLA, a significant correlation was observed between CD36, SRA, ADFP, and IL-8 mRNA levels. Moreover, CD36 expression level was significantly inversely correlated to plasma marker ApoAI.  The above investigated genes/proteins may play a key role in the maturation of atherosclerotic lesions.


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## Body

## 1. Introduction

Macrophage
uptake  of oxidised low density lipoprotein (OxLDL) and  the concomitant
reduction in cholesterol efflux are thought to play a critical role in
atherosclerotic plaque formation and subsequent rupture. A number of macrophage
OxLDL receptors have been identified (e.g.,
CD36, SRBI, SRA) [1, 2] but two in particular, CD36 and SRA,
have been shown to be responsible for up to 90% of OxLDL uptake and degradation
by macrophages [3]. A double knockout mouse model of atherosclerosis
(ApoE−/−, CD36−/−) has many fewer and smaller lesions
compared to ApoE−/− animals that express CD36, even after long-term
feeding with a western diet, [4, 5] which indicates that the CD36
scavenger receptor is implicated in the generation of advanced plaques. This is, however, at variance with
the finding that similar double knockout mice have a comparable, or even
increased, atherosclerotic lesions in the region of the aortic valve [6] as the ApoE−/− mice that have intact CD36, indicating that uptake and endocytosis of OxLDL
seem to be independent of CD36 [6]. Different
possibilities have been recently reviewed to explain such discrepancies [7, 8]. Furthermore,
results from ApoE−/− mice have to be interpreted cautiously, as the
lesions are different from those observed in humans, often with none of the
clinical end points, such as thrombotic ischaemia, resulting in myocardial
infarction or stroke [9]. The
present work has attempted to overcome this limitation by examining human
carotid endarterectomy specimens. Indeed, very few studies have investigated
the expression of CD36 in human vascular lesions [10].Adipophilin
(ADFP), a cellular indicator of the amount of stored lipids, is present in a
very large number of tissues [11]. Increased
expression of ADFP in THP-1 monocytic cell line has been observed to induce cholesterol
storage and reduction of cholesterol efflux [12]. Moreover, its expression in macrophages has been correlated with the presence
of red blood cells and cholesterol crystals in vascular lesions [13]. Finally,
ADFP may play a role in the differentiation of macrophages into foam cells [14].High density lipoproteins
(HDLs) are implicated in the transport of cholesterol to the liver. It is now
well established that there is an inverse relationship between the risk of
developing cardiovascular diseases and the concentration of HDL. However, the
protective effect of HDL is not fully understood. APOAI, the main
apolipoprotein of HDL, plays a critical role in cholesterol efflux from
macrophages and foam cells present in the vessel wall [15].This study investigates genes/proteins implicated in OxLDL uptake
(CD36, SRA), inhibition of cholesterol efflux (ADFP), and inflammatory
recruitments of leukocytes (IL-8) in
plaque lesion areas (PLAs) compared to nonplaque lesion areas (NPLAs) in human carotid
endarterectomy samples. A substantial difference in mRNA and protein
expressions between PLA and NPLA was observed for CD36, ADFP, SRA, and IL-8. In
addition, a significant correlation was obtained between CD36 expression and ADFP,
SRA and IL-8 in PLA. Moreover, a significant inverse correlation was observed
between CD36 gene expression level in PLA and plasma marker ApoAI.


## 2. Materials and Methods

### 2.1. Human Tissue
Samples

Ninety patients,
subjected to endarterectomy (carotid stenosis>70%) at 3 different hospitals
(Red Cross Hospital in Athens (Greece), University Medical Centre in Utrecht (The Netherlands), and Bichat
hospital in Paris (France)),
were enrolled in this study. After removal, endarterectomy samples were
immediately rinsed in RNAse-free sterile PBS, submerged in RNALater (Ambion, Tex, USA),
and stored at −80C°. Ethical committees approved the study, and
patients were fully  informed and gave their written consent prior to enrolment.
Information concerning the patients is summarised in Table 1.Table 1
Patient phenotypes.
n=90Age66.5±8.7Male/Female76%/24%Smokers64%Symptoms56%Diabetes mellitus26.8%Hypertension84%Total cholesterol (mM)4.8±0.9HDl (mM)1.01±0.19LDL (mM)3.1±0.8Triglycerides (mM)3.9±2.3Hematocrit (%)40±4.7White blood cells(10*9/L)7.3±1.4Platelets(10*9/L)218(115–360)Glucose (mg/dL)93±27.6Creatinine (mg/dL)0.9beta-blokers32.8%calcium antagonists30.1%ACE inhibitors42.5%Diuretics29%ARB26%Statins49%Aspirin47.9%Clopidogrel46.2%Insulin3%Diabetes mellitus pills21.9%ApoAI (mg/dL)115±18ApoB (mg/dL)92±23Lp(a)20±48Histological grade (AHA)65.7% grade 628.6% grade 54.3 grade 41.4% grade 3


### 2.2. Endarterectomy
Handling

The lesions were
classified according to the American Heart Association (AHA) criteria [16] by
expert pathologists (Table 1). A transverse (perpendicular to the lumen)
section of the internal carotid artery of 5–10 μm thickness
was taken in the plaque lesion area (PLA) above the bifurcation point (Figure 1).
When available, a macroscopically nonplaque lesion area (NPLA) adjacent to the
atheromatous area, removed as a part of the normal surgical procedure, was used
as an internal control. The PLA and NPLA sections were split horizontally
giving 2 identical pieces of the same area being used, respectively, for
immunohistochemistry and for quantitative PCR (Q-PCR) (Figure 1). Fibroatheroma
(type V) or complicated (type VI) lesions, as described in the AHA criteria
constituted the investigated PLA. Both pieces were washed twice in cold RNAse-free
sterile PBS to remove the RNALater  and either included into OCT or immersed into
RLT lysis buffer (Qiagen, Calif, USA) to protect the RNA and stored at −80C° until further use. For all the samples included into OCT, tissue
sections were cut at 8 μm thickness, dried, and fixed in acetone. Plaque
morphology was also evaluated by staining the tissues with hematoxylin and
eosin.Figure 1
(a) Typical macroscopic
pictures of a carotid endarterectomy specimen. (b) A transverse (perpendicular to the lumen) section of the
internal carotid artery was taken in the plaque lesion area (PLA) above the
bifurcation point. (c) A
macroscopically nonplaque area (NPLA) was used as an internal control. The PLA
and the NPLA sections were split horizontally giving 2 identical pieces that
were used, respectively, for immunohistochemistry and for quantitative PCR.


### 2.3. THP-1 Cells
Culturing and Stimulation

Human monocytic
leukaemia cell line (THP-1/European collection of cell cultures-ECACC no. 88081201)
was cultured in RPMI 1640 (Sigma, Mo, USA) supplemented with 10% heat-inactivated foetal calf
serum (FCS) (Sigma), 1% glutamine (Sigma), 100μg/mL streptomycin
(Sigma) in a humid incubator with 5%CO2 at 37C°. THP-1 cells, cultured in 6-well plates at a
density 1.2×106cells/well, were first treated with 0.2 μM phorbol
12-myristate 13-acetate (PMA,
Sigma) during 24 hours in order to induce the differentiation of monocytes into
macrophages, then washed with sterile PBS and stimulated with 100 μg/mL oxidised low-density lipoproteins (OxLDL, Intracell, USA) during 24 hours. Cell viability was
evaluated using Trypan blue counting to be more than 95% for each assay. Stimulation assays were performed in duplicate and repeated 3 times.


### 2.4. RNA Extraction

THP-1 cells: total RNA
extractions were performed using the RNeasy Mini Kit (Qiagen) following
manufacturer's instructions. RNA quality and quantity were monitored using a Nanodrop spectrophotometer (Nanodrop
Technologies, Del, USA) and an RNA 6000 NanoLabChip kit
on a Bioanalyser (Agilent
Technologies, Calif, USA).Tissue samples: 10 to 30 mg of tissue were used for RNA extraction depending on the
tissue size available. Tissues were first cut into very small pieces and
homogenised with 350 μL of RLT lysis buffer (Qiagen)
using a Polytron (2×30s on ice). After 10 minutes at 55C° proteinase
K step (0.22 g/L−1), the tissue lysate was centrifuged 3 minutes at
10.000 g and RNA extraction was performed on the supernatant following
manufacturer’s instructions using RNeasy Mini Kit (Qiagen). RNA quality and
quantity were monitored using RNA 6000
PicoLabChip/NanoLabChip kit on a Bioanalyser (Agilent) and using the Nanodrop spectrophotometer
(Nanodrop Technologies). Total RNA extracted from highly calcified lesions was
poor and these were removed from the cohort.


### 2.5. Retrotranscription (RT) and Real-Time Quantitative PCR (Q-PCR)

Only total RNA extracts presenting
good integrity on Bioanalyser profiles were retrotranscribed
(1μg for THP-1 cells or 200 ng for tissue samples) with superscript III reverse
transcriptase (Invitrogen) following manufacturer’s instructions.Q-PCR assays were carried out using the 7900 HT Sequence Detector
System (Applied Biosystems/Ambion,
(AB)). Five microlitres of 10X RT product were amplified using 1X of Universal
PCR Master Mix (AB), 1X assay-on-demand (containing specific primers and
labelled probe, AB) in a final volume of 25μL. Q-PCR amplification
was performed in duplicate. 18S rRNA (AB) amplification was used for
normalisation. Each gene was assayed individually.


### 2.6. Immunohistochemistry and
Quantitative Image Analysis

Serial sections of endarterectomy
samples (NPLA and PLA) were first immunostained with an anti-CD68 (1/500, Dako,
Glostrup, Denmark), an antialpha actin (1/50, Dako),
and an anti-CD31 (1/20, Dako) in order to localise macrophages, smooth muscle
cells (SMCs), and endothelial cells, respectively. Then sections were incubated
with either an anti-CD36 monoclonal antibody (1/25, BioCytex, Marseille, France),  an anti-SRA (1/100, R&D Systems, Minn, USA), an anti-ADFP (1/200, Chemicon, Millipore, Billerica, USA), or the corresponding IgG isotype. Staining was visualised using the Envision kit
(Dako). Slides were then counterstained with hematoxylin and mounted in DPX (Sigma). Staining of CD36,
SRA, and ADFP was quantitatively analysed by the use of a colour image analyser (Sight System,
Newbury, Berks, UK;
Software by Foster-Finley Associates, Newcastle, UK) detecting the peroxidase
reaction product by measurement of pixels in terms of hue, saturation, and
intensity values. By this method, the percentage of the stained tissue was
measured. For each tissue, the entire section was analysed and the intensity
staining obtained was normalised
via the surface of the tissue analysed [17, 18].


### 2.7. Western-Blot

Proteins were extracted using
5%-tritonX-100 lysis buffer. Twenty micrograms of total proteins were subjected
to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The
membranes were incubated with an anti-CD36 monoclonal antibody (Clone 10/5) (BioCytex),
or with an anti-ADFP polyclonal antibody (AB9102) (Chemicon) at dilutions 1/100
and 1/2000, respectively. Detection was performed using ECL western blotting
detection reagents (Amersham, Buckinghamshire, UK) and exposure to Kodak Biomax XAR
films (Sigma). Signals were analysed and compared using GelDoc software (BioRad
Laboratories, Calif, USA).


### 2.8. ELISA

IL-8  levels were  measured in cell supernatants in duplicate using an
IL-8 ELISA Kit (Quantikine from R&D Systems) accordingto the
supplier’s instructions.


### 2.9. Statistical Analysis

Statistical analyses were performed using
Wilcoxon matched pairs tests
(ranks test) and
Mann-Whitney tests (Statistica software, Statsoft). Online software (http://www.fon.hum.uva.nl/Service/Statistics/Correlation_coefficient.html) was
used to conduct Pearson’s correlation tests.
In all cases, a value of P≤.05 was regarded as significant.


## 2.1. Human Tissue
Samples

Ninety patients,
subjected to endarterectomy (carotid stenosis>70%) at 3 different hospitals
(Red Cross Hospital in Athens (Greece), University Medical Centre in Utrecht (The Netherlands), and Bichat
hospital in Paris (France)),
were enrolled in this study. After removal, endarterectomy samples were
immediately rinsed in RNAse-free sterile PBS, submerged in RNALater (Ambion, Tex, USA),
and stored at −80C°. Ethical committees approved the study, and
patients were fully  informed and gave their written consent prior to enrolment.
Information concerning the patients is summarised in Table 1.Table 1
Patient phenotypes.
n=90Age66.5±8.7Male/Female76%/24%Smokers64%Symptoms56%Diabetes mellitus26.8%Hypertension84%Total cholesterol (mM)4.8±0.9HDl (mM)1.01±0.19LDL (mM)3.1±0.8Triglycerides (mM)3.9±2.3Hematocrit (%)40±4.7White blood cells(10*9/L)7.3±1.4Platelets(10*9/L)218(115–360)Glucose (mg/dL)93±27.6Creatinine (mg/dL)0.9beta-blokers32.8%calcium antagonists30.1%ACE inhibitors42.5%Diuretics29%ARB26%Statins49%Aspirin47.9%Clopidogrel46.2%Insulin3%Diabetes mellitus pills21.9%ApoAI (mg/dL)115±18ApoB (mg/dL)92±23Lp(a)20±48Histological grade (AHA)65.7% grade 628.6% grade 54.3 grade 41.4% grade 3


## 2.2. Endarterectomy
Handling

The lesions were
classified according to the American Heart Association (AHA) criteria [16] by
expert pathologists (Table 1). A transverse (perpendicular to the lumen)
section of the internal carotid artery of 5–10 μm thickness
was taken in the plaque lesion area (PLA) above the bifurcation point (Figure 1).
When available, a macroscopically nonplaque lesion area (NPLA) adjacent to the
atheromatous area, removed as a part of the normal surgical procedure, was used
as an internal control. The PLA and NPLA sections were split horizontally
giving 2 identical pieces of the same area being used, respectively, for
immunohistochemistry and for quantitative PCR (Q-PCR) (Figure 1). Fibroatheroma
(type V) or complicated (type VI) lesions, as described in the AHA criteria
constituted the investigated PLA. Both pieces were washed twice in cold RNAse-free
sterile PBS to remove the RNALater  and either included into OCT or immersed into
RLT lysis buffer (Qiagen, Calif, USA) to protect the RNA and stored at −80C° until further use. For all the samples included into OCT, tissue
sections were cut at 8 μm thickness, dried, and fixed in acetone. Plaque
morphology was also evaluated by staining the tissues with hematoxylin and
eosin.Figure 1
(a) Typical macroscopic
pictures of a carotid endarterectomy specimen. (b) A transverse (perpendicular to the lumen) section of the
internal carotid artery was taken in the plaque lesion area (PLA) above the
bifurcation point. (c) A
macroscopically nonplaque area (NPLA) was used as an internal control. The PLA
and the NPLA sections were split horizontally giving 2 identical pieces that
were used, respectively, for immunohistochemistry and for quantitative PCR.


## 2.3. THP-1 Cells
Culturing and Stimulation

Human monocytic
leukaemia cell line (THP-1/European collection of cell cultures-ECACC no. 88081201)
was cultured in RPMI 1640 (Sigma, Mo, USA) supplemented with 10% heat-inactivated foetal calf
serum (FCS) (Sigma), 1% glutamine (Sigma), 100μg/mL streptomycin
(Sigma) in a humid incubator with 5%CO2 at 37C°. THP-1 cells, cultured in 6-well plates at a
density 1.2×106cells/well, were first treated with 0.2 μM phorbol
12-myristate 13-acetate (PMA,
Sigma) during 24 hours in order to induce the differentiation of monocytes into
macrophages, then washed with sterile PBS and stimulated with 100 μg/mL oxidised low-density lipoproteins (OxLDL, Intracell, USA) during 24 hours. Cell viability was
evaluated using Trypan blue counting to be more than 95% for each assay. Stimulation assays were performed in duplicate and repeated 3 times.


## 2.4. RNA Extraction

THP-1 cells: total RNA
extractions were performed using the RNeasy Mini Kit (Qiagen) following
manufacturer's instructions. RNA quality and quantity were monitored using a Nanodrop spectrophotometer (Nanodrop
Technologies, Del, USA) and an RNA 6000 NanoLabChip kit
on a Bioanalyser (Agilent
Technologies, Calif, USA).Tissue samples: 10 to 30 mg of tissue were used for RNA extraction depending on the
tissue size available. Tissues were first cut into very small pieces and
homogenised with 350 μL of RLT lysis buffer (Qiagen)
using a Polytron (2×30s on ice). After 10 minutes at 55C° proteinase
K step (0.22 g/L−1), the tissue lysate was centrifuged 3 minutes at
10.000 g and RNA extraction was performed on the supernatant following
manufacturer’s instructions using RNeasy Mini Kit (Qiagen). RNA quality and
quantity were monitored using RNA 6000
PicoLabChip/NanoLabChip kit on a Bioanalyser (Agilent) and using the Nanodrop spectrophotometer
(Nanodrop Technologies). Total RNA extracted from highly calcified lesions was
poor and these were removed from the cohort.


## 2.5. Retrotranscription (RT) and Real-Time Quantitative PCR (Q-PCR)

Only total RNA extracts presenting
good integrity on Bioanalyser profiles were retrotranscribed
(1μg for THP-1 cells or 200 ng for tissue samples) with superscript III reverse
transcriptase (Invitrogen) following manufacturer’s instructions.Q-PCR assays were carried out using the 7900 HT Sequence Detector
System (Applied Biosystems/Ambion,
(AB)). Five microlitres of 10X RT product were amplified using 1X of Universal
PCR Master Mix (AB), 1X assay-on-demand (containing specific primers and
labelled probe, AB) in a final volume of 25μL. Q-PCR amplification
was performed in duplicate. 18S rRNA (AB) amplification was used for
normalisation. Each gene was assayed individually.


## 2.6. Immunohistochemistry and
Quantitative Image Analysis

Serial sections of endarterectomy
samples (NPLA and PLA) were first immunostained with an anti-CD68 (1/500, Dako,
Glostrup, Denmark), an antialpha actin (1/50, Dako),
and an anti-CD31 (1/20, Dako) in order to localise macrophages, smooth muscle
cells (SMCs), and endothelial cells, respectively. Then sections were incubated
with either an anti-CD36 monoclonal antibody (1/25, BioCytex, Marseille, France),  an anti-SRA (1/100, R&D Systems, Minn, USA), an anti-ADFP (1/200, Chemicon, Millipore, Billerica, USA), or the corresponding IgG isotype. Staining was visualised using the Envision kit
(Dako). Slides were then counterstained with hematoxylin and mounted in DPX (Sigma). Staining of CD36,
SRA, and ADFP was quantitatively analysed by the use of a colour image analyser (Sight System,
Newbury, Berks, UK;
Software by Foster-Finley Associates, Newcastle, UK) detecting the peroxidase
reaction product by measurement of pixels in terms of hue, saturation, and
intensity values. By this method, the percentage of the stained tissue was
measured. For each tissue, the entire section was analysed and the intensity
staining obtained was normalised
via the surface of the tissue analysed [17, 18].


## 2.7. Western-Blot

Proteins were extracted using
5%-tritonX-100 lysis buffer. Twenty micrograms of total proteins were subjected
to SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes. The
membranes were incubated with an anti-CD36 monoclonal antibody (Clone 10/5) (BioCytex),
or with an anti-ADFP polyclonal antibody (AB9102) (Chemicon) at dilutions 1/100
and 1/2000, respectively. Detection was performed using ECL western blotting
detection reagents (Amersham, Buckinghamshire, UK) and exposure to Kodak Biomax XAR
films (Sigma). Signals were analysed and compared using GelDoc software (BioRad
Laboratories, Calif, USA).


## 2.8. ELISA

IL-8  levels were  measured in cell supernatants in duplicate using an
IL-8 ELISA Kit (Quantikine from R&D Systems) accordingto the
supplier’s instructions.


## 2.9. Statistical Analysis

Statistical analyses were performed using
Wilcoxon matched pairs tests
(ranks test) and
Mann-Whitney tests (Statistica software, Statsoft). Online software (http://www.fon.hum.uva.nl/Service/Statistics/Correlation_coefficient.html) was
used to conduct Pearson’s correlation tests.
In all cases, a value of P≤.05 was regarded as significant.


## 3. Results

### 3.1. OxLDL stimulation in PMA-differentiated THP-1 macrophages

An in vitro approach was first used to
investigate the gene and protein expressions, under our laboratory conditions, of 4 OxLDL-targeted
genes (CD36, SRA, ADFP, IL-8) prior to starting the work on endarterectomy
tissues. Twenty four hours of OxLDL treatment (100μg/mL) of PMA-differentiated THP-1 macrophages, compared to cells incubated with PBS
only, induced a significant increase of CD36 (4.1±1.1-fold), IL-8 (3.4±0.9-fold),
and ADFP (7.4±1.4-fold) gene expressions, P<.05 (Figure 2(a)). In contrast, a decrease of SRA gene expression (0.5±0.1-fold)
was observed in these cells after OxLDL treatment (100 μg/mL) P<.05. Western-blot analysis
showed that CD36 and ADFP protein levels were also increased respectively by 132% (±30%) (P = .05) and by 31% (±3%, P=.01) after the OxLDL
treatment (Figures 2(b) and 2(c)). IL-8  increased by 22% (±1.1%, P=.005)
in cell  supernatant using  ELISA  assays (Figure 2(d)).Comparison of gene/protein expression patterns between
OxLDL-treated (100μg/mL, for 24 hours) and
PBS-treated PMA-differentiated THP-1 cells. (a) Real-time quantitative PCR showing gene expression levels for
CD36, SRA, adipophilin (ADFP), and IL-8. (b),
(c) Western-blot analysis of, respectively, CD36 and ADFP protein expression
levels. (d) IL-8 protein levels in
THP-1 culture supernatants assayed by ELISA. Treated and untreated cells are,
respectively, represented by plain and dotted bars. Bars represent the means ±
SE for 3 different experiments performed in duplicate. Wilcoxon matched pairs
test was used to determine significance.      P≤.01**,P≤.05*.


### 3.2. Comparison of Gene and Protein Expressions in PLA Versus NPLA

Gene and protein expressions
(CD36, SRA, ADFP, and IL-8) at sites of PLA were compared to NPLA in
endarterectomy samples. CD36 (6.80 versus 5.41,P=.0045), ADFP (6.85 versus
5.78, P=.0017), SRA (6.93 versus 6.27, P=.23), and IL-8 (5.98
versus 4.93, P=.0065) genes were shown to be significantly more
expressed in PLA compared to NPLA
in the 15 patient’s samples investigated (Figure 3(a)). Furthermore, PLA showed significantly higher CD36 (0.15 versus
0.019, P=.017), ADFP (0.051 versus 0.026, P=.049), and SRA (0.081
versus 0.027, P=.027) protein levels compared to NPLA using
quantitative immunostaining techniques in the 8 patient’s samples investigated (Figures
3(b), 3(c), and 3(d)).Gene and protein expression levels in carotid endarterectomy PLAs
(plaque lesion areas) and NPLAs (nonplaque lesion areas). (a) Real-time quantitative PCR showing gene expression levels for
CD36, adipophilin (ADFP), SRA, and IL-8 in carotid endarterectomy PLA compared
to NPLA. (b) Staining of negative
isotype, CD68,α-actin, and CD36
proteins in PLA and NPLA and quantitative immunohistochemistry for CD36 protein
expression in PLA versus NPLA. (c)
Staining of negative isotype, CD68, α-actin, and ADFP
proteins in PLA and NPLA and quantitative immunohistochemistry for ADFP protein
expression in PLA versus NPLA. (d)
Staining of negative isotype, CD68, α-actin, and SRA
proteins in PLA and NPLA and quantitative immunohistochemistry for SRA protein
expression in PLA versus NPLA. Wilcoxon matched pairs test was used to
determine significance.      P≤.001**,P≤.05*; L: lumen; magnification: 10X.
(a)(b)(c)(d)


### 3.3. Correlation
of CD36 with SRA or ADFP in Carotid Endarterectomy Specimens

In PLA samples, a significant correlation was observed between CD36 and
SRA mRNA expression levels(n=72,R=.767,P<.01). Interestingly,
a significant correlation was also observed between CD36 and ADFP (n=88,R=.786,P<.01) and between CD36 and IL-8 (n=88,R=.499,P<.01) mRNA expression levels (Figure 4).Correlation of CD36 with (a)
SRA(n=72), (b) adipophilin (n=88), or (c) IL-8 (n=88) gene
expression in plaque lesion area (PLA). (d) Correlation of CD36 gene
expression in plaque lesion areas (PLAs) with plasma marker ApoAI level in 57
patients. Pearson’s correlation statistical test was used.
(a)(b)(c)(d)


### 3.4. Correlation between CD36
with ApoAI and HDL

A significant inverse correlation was
observed between CD36 gene expression level in PLA samples(n=57) and plasma
marker ApoAI (R=.288,P=.029) (Figure 4(d)). Furthermore, an
inverse correlation was also found between CD36 gene expression and HDL plasma
level, however it was not statistically significant (R=.207,P=.1). No correlation was observed between CD36 and total cholesterol, LDL,
triglycerides, plasma ApoB, medication, smoking status or gender (data not
shown).


## 3.1. OxLDL stimulation in PMA-differentiated THP-1 macrophages

An in vitro approach was first used to
investigate the gene and protein expressions, under our laboratory conditions, of 4 OxLDL-targeted
genes (CD36, SRA, ADFP, IL-8) prior to starting the work on endarterectomy
tissues. Twenty four hours of OxLDL treatment (100μg/mL) of PMA-differentiated THP-1 macrophages, compared to cells incubated with PBS
only, induced a significant increase of CD36 (4.1±1.1-fold), IL-8 (3.4±0.9-fold),
and ADFP (7.4±1.4-fold) gene expressions, P<.05 (Figure 2(a)). In contrast, a decrease of SRA gene expression (0.5±0.1-fold)
was observed in these cells after OxLDL treatment (100 μg/mL) P<.05. Western-blot analysis
showed that CD36 and ADFP protein levels were also increased respectively by 132% (±30%) (P = .05) and by 31% (±3%, P=.01) after the OxLDL
treatment (Figures 2(b) and 2(c)). IL-8  increased by 22% (±1.1%, P=.005)
in cell  supernatant using  ELISA  assays (Figure 2(d)).Comparison of gene/protein expression patterns between
OxLDL-treated (100μg/mL, for 24 hours) and
PBS-treated PMA-differentiated THP-1 cells. (a) Real-time quantitative PCR showing gene expression levels for
CD36, SRA, adipophilin (ADFP), and IL-8. (b),
(c) Western-blot analysis of, respectively, CD36 and ADFP protein expression
levels. (d) IL-8 protein levels in
THP-1 culture supernatants assayed by ELISA. Treated and untreated cells are,
respectively, represented by plain and dotted bars. Bars represent the means ±
SE for 3 different experiments performed in duplicate. Wilcoxon matched pairs
test was used to determine significance.      P≤.01**,P≤.05*.


## 3.2. Comparison of Gene and Protein Expressions in PLA Versus NPLA

Gene and protein expressions
(CD36, SRA, ADFP, and IL-8) at sites of PLA were compared to NPLA in
endarterectomy samples. CD36 (6.80 versus 5.41,P=.0045), ADFP (6.85 versus
5.78, P=.0017), SRA (6.93 versus 6.27, P=.23), and IL-8 (5.98
versus 4.93, P=.0065) genes were shown to be significantly more
expressed in PLA compared to NPLA
in the 15 patient’s samples investigated (Figure 3(a)). Furthermore, PLA showed significantly higher CD36 (0.15 versus
0.019, P=.017), ADFP (0.051 versus 0.026, P=.049), and SRA (0.081
versus 0.027, P=.027) protein levels compared to NPLA using
quantitative immunostaining techniques in the 8 patient’s samples investigated (Figures
3(b), 3(c), and 3(d)).Gene and protein expression levels in carotid endarterectomy PLAs
(plaque lesion areas) and NPLAs (nonplaque lesion areas). (a) Real-time quantitative PCR showing gene expression levels for
CD36, adipophilin (ADFP), SRA, and IL-8 in carotid endarterectomy PLA compared
to NPLA. (b) Staining of negative
isotype, CD68,α-actin, and CD36
proteins in PLA and NPLA and quantitative immunohistochemistry for CD36 protein
expression in PLA versus NPLA. (c)
Staining of negative isotype, CD68, α-actin, and ADFP
proteins in PLA and NPLA and quantitative immunohistochemistry for ADFP protein
expression in PLA versus NPLA. (d)
Staining of negative isotype, CD68, α-actin, and SRA
proteins in PLA and NPLA and quantitative immunohistochemistry for SRA protein
expression in PLA versus NPLA. Wilcoxon matched pairs test was used to
determine significance.      P≤.001**,P≤.05*; L: lumen; magnification: 10X.
(a)(b)(c)(d)


## 3.3. Correlation
of CD36 with SRA or ADFP in Carotid Endarterectomy Specimens

In PLA samples, a significant correlation was observed between CD36 and
SRA mRNA expression levels(n=72,R=.767,P<.01). Interestingly,
a significant correlation was also observed between CD36 and ADFP (n=88,R=.786,P<.01) and between CD36 and IL-8 (n=88,R=.499,P<.01) mRNA expression levels (Figure 4).Correlation of CD36 with (a)
SRA(n=72), (b) adipophilin (n=88), or (c) IL-8 (n=88) gene
expression in plaque lesion area (PLA). (d) Correlation of CD36 gene
expression in plaque lesion areas (PLAs) with plasma marker ApoAI level in 57
patients. Pearson’s correlation statistical test was used.
(a)(b)(c)(d)


## 3.4. Correlation between CD36
with ApoAI and HDL

A significant inverse correlation was
observed between CD36 gene expression level in PLA samples(n=57) and plasma
marker ApoAI (R=.288,P=.029) (Figure 4(d)). Furthermore, an
inverse correlation was also found between CD36 gene expression and HDL plasma
level, however it was not statistically significant (R=.207,P=.1). No correlation was observed between CD36 and total cholesterol, LDL,
triglycerides, plasma ApoB, medication, smoking status or gender (data not
shown).


## 4. Discussion

In this study, a substantial difference in mRNA and protein expressions was observed
for CD36 and molecules implicated in lipid metabolism (ADFP, SRA) and
inflammation (IL-8) between plaque lesion area (PLA) and nonplaque lesion area
(NPLA) in human carotid endarterectomies. Moreover, results obtained for an in vitro
macrophage model (PMA-treated THP-1 cells) stimulated by OxLDL show a trend,
with the exception of SRA, that is similar to those obtained in PLA. In
addition, a significant correlation was obtained between CD36 expression and ADFP,
SRA, and IL-8 in PLA. Furthermore, a significant inverse correlation was
observed between CD36 gene expression level in PLA and plasma marker ApoAI.CD36,  ADFP, and
SRA are strongly correlated in this study in PLA and are potentially proatherogenic
proteins. Indeed, resident vascular macrophages have scavenger receptors CD36
and SRA that play a key role in the uptake of OxLDL. Moreover, ADFP in these
very same macrophages is involved in lipid accumulation by inhibiting efflux [12]. It has also been described that the
ABCA1 transporter protein expression, involved in the cholesterol transport
from cells to apolipoproteins in plasma, was significantly reduced in carotid
lesions compared with controls tissues [19]. The uptake of
OxLDL upregulates scavenger receptors and the adipophilin expression and
ensures the formation of foam cells and the perpetuation of lesions. An inflammatory component (IL-8) that is
correlated to CD36 in PLA is also observed. This brings a further dimension to
the capacity of resident macrophages to attract further monocytes to the sites
of inflammation through the release of IL-8. An additional interesting finding
in this study is that the plasma acceptor ApoAI, a constituent of HDL, needed
for transportation of cholesterol from macrophages is significantly inversely correlated to CD36 gene expression level in PLA. Macrophages
do not synthesise ApoAI and need to have it readily available in plasma as a
cholesterol acceptor. In the absence of ApoAI, cholesterol efflux is greatly
reduced. HDL levels were also inversely correlated to CD36 gene expression but this
correlation did not reach statistical significance.CD36 in this
study is statistically more highly expressed in PLA (grade V and VI lesions,
AHA classifications) [16] compared
to NPLA regions. Cells expressing CD36 are mostly macrophages as indicated by
CD68 labelling. CD36 in murine ApoE−/− macrophages, compared to ApoE−/− CD36−/−, has been shown in several studies to be linked to the generation
of atherosclerotic lesions. However, a certain caution needs to be taken in
trying to understand human atherosclerotic lesions compared to those generated
in ApoE−/− mice. Indeed, in the ApoE−/− murine model of
atherosclerosis there is an absence of myocardial infarction or stroke that are
the results of clinical complications of atherosclerosis in patients. The
intense CD36 labelling obtained in this study for PLA compared to NPLA suggests
that this scavenger receptor is actively involved in the perpetuation of
lesions in human vessels.The PMA-differentiated THP-1 incubated with OxLDL
showed a similar upregulation of CD36, ADFP, and IL-8 gene and protein
expressions compared to those obtained in PLA versus NPLA. In contrast, SRA
which is enhanced in PLA versus NPLA was shown to be decreased by OxLDL
treatment in PMA-differentiated THP-1. It is
conceivable that SRA and CD36 might be regulated differentially and therefore
could play different roles in the formation of foam cells in atherosclerotic
lesions. Indeed, CD36 and SRA are differently expressed in lesion area present
in post-mortem descending human thoracic aorta [20]. Alternatively,
a monocytic cell line such as THP-1 cells may differ in its response from
normal monocytes/macrophages.A certain caution needs to be taken in
interpreting the above data. However, an interesting working hypothesis would
rely on carotid resident macrophages to take up OxLDL via upregulated CD36 and
SRA and inhibit cholesterol efflux as a result of the high expression of ADFP
and low levels of plasma cholesterol acceptor ApoAI. Moreover, IL-8 would be
implicated in monocyte recruitment to the sites of inflammation (Figure5).Figure 5
Hypothetical representation of OxLDL influx and inhibition of
cholesterol efflux in carotid resident macrophages. In the presence of high
expression of CD36 in plaque lesion areas, the observed strong correlation
between CD36, SRA, and ADFP suggests that carotid lesion resident macrophages
are subjected on the one hand to greatly enhanced OxLDL uptake by CD36 and SRA
and on the other to inhibition of cholesterol efflux by ADFP. The cholesterol
efflux from resident macrophages is probably even further reduced as indicated
by the inverse correlation of CD36 expression with plasma cholesterol acceptor
ApoAI.



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*Source: 813236-2008-09-28.xml*