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)

*** ***