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Anil Paul Chirackal Manavalan1, Alexandra Kober1, Jari Metso2, Tatjana Becker1, Karin Hasslitzer1, Cornelia Schweinzer1,
Jasminka Stefulj3, Matti Jauhiainen2, and Ute Panzenboeck1
1Institute of Pathophysiology and Immunology, Medical University of Graz, Austria; 2National Institute for Health and Welfare, Biomedicum, Helsinki, Finland; 3Department of Molecular Biology, Ruder
Boskovic Institute, Zagreb, Croatia
Phospholipid transfer protein is expressed in
cerebrovascular endothelial cells and involved in HDL
genesis at the blood brain barrier
INTRODUCTION / OBJECTIVES RESULTS
SUMMARY/CONCLUSION
We report for the first time that pBCEC express and secrete significant amounts of PLTP (Fig. 1A), and its mRNA expression levels (Fig. 1B) and
activity (Fig. 2B) can be enhanced by LXR activation using endogenous (24-OH Chol) or synthetic (TO901317) ligands. Two-dimensional
immunoelectrophoresis revealed that pre-β-HDL is formed by pBCEC upon incubation with human HDL3 and there is an increased trend in pre-β-HDL
formation with LXR stimulation (Fig. 3A &B) which is in line with increased PLTP activity (Fig. 3C). Pre-incubation with isolated active plasma PLTP
enhanced the capacity of HDL3 to efflux cholesterol from pBCEC in a time dependent manner (Fig. 4). Furthermore, RNAi mediated PLTP silencing
revealed that PLTP contributes to both apoA-I and HDL3 mediated cholesterol efflux from pBCEC (Fig. 5). Based on our current findings we propose
that PLTP may represent a key player in HDL genesis/ remodeling at the BBB and in lipid transport between the brain and the circulation.
REFERENCES
1, Huuskonen J et al. 2001 Athero 155:269-81
2, Tzotzas et al. 2009 Obes Rev 10:403-11
3, Riemens SC et al. 1998 Diabetologia 41:929-34
4, Vuletic S et al. 2003 J Lipid Res 44:1113-23
5, Panzenboeck U et al. 2002 J Biol Chem 277:42881-89
6, Desrumaux C et al. 2001 J Biol Chem 276: 5908-5915
7, Marik J et al. 2007 Nucl Med Biol 34(2):165-71.
Phospholipid transfer protein (PLTP) facilitates the exchange of phospholipids (PL),
unesterified cholesterol, and vitamin E among various lipoproteins as well as
between lipoproteins and cells. In the periphery, PLTP plays a significant role in
remodeling of plasma high density lipoproteins (HDL), converting them into
populations of larger and smaller particles, both representing efficient acceptors of
cellular cholesterol.1 Both pro- or anti-atherogenic effects of PLTP have been
reported, and elevated plasma PLTP activity in insulin resistance is associated with
obesity.2,3 In Alzheimer‘s disease (AD), levels of brain parenchymal PLTP are
increased but PLTP activity in cerebrospinal fluid is decreased.4 However, the
potential functions of PLTP in the CNS have not been described. We previously
reported that liver X receptor (LXR) activation promotes cellular cholesterol efflux
and formation of HDL-like particles in an established in vitro model of the blood-
brain barrier (BBB) consisting of porcine brain capillary endothelial cells (pBCEC)5.
We here investigated the expression, regulation, and function of PLTP, another LXR
target, in pBCEC.
0
50
100
150
200
250
Control 24OH-C TO
PC
tra
nsp
ort
(n
mo
l/mg
/ml/h
)
Media
cell lysates
*
* *
(B)
0
200
400
Control 24OH-C TO
mR
NA
(P
LTP
/HP
RT
1)
* *
(B)
Fig. 2: PLTP activity is elevated upon LXR activation. (A) Scheme of PLTP activity assay (3H-
DPPC tritium labeled dipalmitoylphosphatidylcholine). (B) pBCEC were cultured and treated as
described in the legend of Fig. 1. PLTP activity of media and cell lysates were determined based on
the transfer of [³H]-PC from liposomes to human HDL3. Values were normalized to total cellular
protein contents. (Means ± SE of 3 independent experiments, n = 3, * p< 0.05 vs controls)
KDa
Secreted PLTP
C C C 24 24 24 TO TO TO
ß-Actin
Intracelluar PLTP
~55
~55
~42
Fig. 1: PLTP is secreted, expressed and regulated by LXR activation in pBCEC. Cerebrovascular
endothelial cells were isolated from porcine brains and cultured on 6-well plates after a single passage.
Confluent pBCEC were incubated in the presence or absence of LXR ligands 24-OH Chol (10 µM) or
TO901317 (5 µM) for 24 h in serum-free medium. (A) Proteins were extracted from cells and TCA-
precipitated from media, separated by SDS-PAGE (4-12 %) and PLTP was immunodetected using
rabbit polyclonal anti-PLTP antibody. Band intensities were evaluated by densitometric scanning. (B)
RNA was isolated, reversed transcribed and real-time PCR was performed using SYBR Green
technology. mRNA expression levels were normalized to HPRT1. (Means ± SE of 3 independent
experiments, n = 3, * p <0.05, ** p< 0.01, *** p< 0.001 vs controls)
Fig. 4: Exogenous PLTP enhances the cholesterol efflux capacity of HDL3. pBCEC were
cultured on 12-well plates, labeled with [3H]-cholesterol (0.5 µCi/ml) for 24 h in the absence (A) or
presence of 24-OH Chol (10 µM; B) or TO901317 (5 µM; C). Cellular cholesterol pools were
equilibrated for 16 h and time-dependent cellular [3H]-cholesterol efflux to HDL3 (50 µg/ml) was
determined after pre-incubation of HDL3 in the absence (PBS) or presence of plasma PLTP (1000
nmol/h for 100 µg HDL3 , 37oC, 24 h) . [Means ± SE of 2 independent experiments , n = 3,
≠ p< 0.0001 vs without PLTP pre-treatment, ***p 0.001 vs unstimulated cells (A)]
Fig. 5. PLTP silencing reduces both apoA-I and HDL3 mediated cholesterol efflux from pBCEC.
pBCEC were cultured on 12-well plates. Cells were labeled with [3H]-cholesterol (0.5 µCi/ml) and
simultaneously transfected with hPLTP siRNA, hABCG1 or both (25 nM) for 48 h. Scrambled
oligonucleotides were used as the negative control. Cellular cholesterol pools were equilibrated for 2 h
and time-dependent cellular [3H]-cholesterol efflux to apoA-I (A; 10 µg/ml) or HDL3 (B; 50 µg/ml) were
determined. (C) RNA was isolated, reversed transcribed and real-time PCR was performed using
SYBR Green technology. mRNA expression levels were normalized to HPRT1. (Means ± SD of
one experiment representative of 3, n = 3, * p< 0.05, ** p< 0.01, *** p< 0.001 vs scrambled)
(A)
3H-DPPC
HDL3
PLTP
(A)
***
(B)
Modified from Desrumaux et al. 2001
& Marik et al. 2007
PLTP mRNA levels PLTP protein levels
PLTP activity
(A) (B) (C)
0
1
2
3
4
5
6
7
8
9
10
1.5 h 4 h 24 h
% T
ota
l [3 H
]-C
ho
lest
ero
l
ApoA-I mediated efflux
Scrambled
si PLTP
*
**
**
(A)
0
5
10
15
20
25
1.5 h 4 h 24 h
% T
ota
l [3 H
]-C
ho
lest
ero
l
HDL3 mediated efflux
Scrambled
si PLTP
si ABCG 1
si P + G1
* *
* * *
* * *
,00
,400
,800
1,200
mR
NA
(P
LTP
/HP
RT
1)
PLTP expression
levels
***
(C)
Fig. 3: Pre-β-HDL is formed and improved upon LXR activation in pBCEC. pBCEC were cultured on
75 cm2 flasks and incubated in the absence or presence of TO 901317 (5 µM) and/or HDL3 (50 µg/ml)
for 24 h in serum-free medium. Media was collected and concentrated using Amicon Ultra-10K
centrifugal fiters . Amounts of pre-β-HDL and α-HDL were measured by two-dimensional crossed
immunoelectrophoresis (A). (B) The amounts of pre-β-HDL are expressed as percentages of the sum
of α-HDL and pre-β-HDL. (C) PLTP activity of the media was also determined as described in the
legend of Fig. 2 (B). (Means ± SD of one experiment representative of 2 n = 3).
,00
10,00
20,00
30,00
pBCEC+ HDL3 pBCEC+TO+ HDL3
% p
re-β
-H
DL
% Pre-β-HDL
0
400
800
1200
pBCEC+ HDL3 pBCEC+TO+ HDL3
PC
tra
nsp
ort
(n
mo
l/mg
/ml/h
)
PLTP activity pBCEC+ HDL3 pBCEC+TO+HDL3
Control HDL3; 370C Control HDL3; 40C
Preβ
α (A) (B)
(C)
*** ***