Atherosclerosis 123 (1996) I - I5

atherosclerosis

Review article

Transfer of low density lipoprotein into the arterial wall and risk of atherosclerosis

Lars Bo Nielsen* Depnrtment oj Cbnical Biochemistry, Rigshospitalet, Unitwrsity of Copenhagen, Copenhagen, Denmark

Received 19 October 1995; revised 8 January 1996; accepted 8 January 1996

Abstract

The aim of the review is to summarize the present knowledge on determinants of transfer of low density lipoprotein (LDL) into the arterial wall, particularly in relation to the risk of development of atherosclerosis. The flux of LDL into the arterial wall (in moles of LDL per surface area per unit of time) has two major determinants, i.e. the LDL concentration in plasma and the arterial wall permeability. LDL enters the arterial wall as intact particles by vesicular ferrying through endothelial cells and/or by passive sieving through pores in or between endothelial cells. Estimates in vivo of the LDL permeability of a normal arterial wall vary between 5 and 100 nl/cm’/h. In laboratory animals, the regional variation in the arterial wall permeability predicts the pattern of subscqucnt dietary induced atheroscle- rosis. Moreover, mechanical or immunological injury of the arterial wall increases the LDL permeability and is accompanied by accelerated development of experimental atherosclerosis. This supports the idea that an increased permeability to LDL, like an increased plasma LDL concentration, increases the risk of atherosclerosis. Hyperten- sion, smoking, genetic predisposition, atherosclerosis, and a small size of LDL may all increase the arterial wall permeability to LDL and in this way increase the risk of accelerated development of atherosclerosis. The hypothesis that atherosclerosis risk can be reduced by improving the barrier function of the arterial wall towards the entry of LDL remains to be investigated; agents which directly modulate the LDL permeability of the arterial wall in vivo await identification.

Keywords: Atherosclerosis; Endothelium; Lipoprotein kinetics; Low density lipoprotein; Permeability

1. Introduction

The accumulation of atherogenic lipoproteins in the arterial wall intima constitutes a fimdamen- tal event in atherogenesis [1,2]; low density lipo- protein is the most abundant atherogenic lipoprotein in plasma and high plasma levels of

* Current address: Gladstone Institute of Cardiovascular Disease, University of California, San Francisco 2550 23rd St., PO Box 419100, San Francisco, CA 94141-9100, USA. Tel.: + 1 415 826 7500: fax: + 1 415 285 5632; e-mail: Lars-Bo- Nielsen@quickmail.ucsf.edu

0021-9150/96;$15.00 Q 1996 Elsevier Science Ireland Ltd. All rights reserved PII SOO21-9150(96)05802-9

2 L.B. Nielsen 1 Atherosclerosis 123 (1996) l-15

LDL are causally related to the development of atherosclerosis [3]. It seems important therefore to understand factors determining transport of LDL into the arterial wall. The aim of this review is to summarize the present knowledge on determi- nants of transfer of LDL into the arterial wall, particularly in relation to the risk of developing atherosclerosis.

2. Plasma concentration of LDL and arterial wall permeability are independent determinants of the transfer of LDL into the arterial wall

Studies in humans, pigeons and rabbits indicate that the flux of LDL from plasma into the arterial wall (in moles of LDL per unit surface area per unit of time) depends on the plasma concentration of LDL as well as LDL permeability at the plasma-arterial wall interface [4-61. The influx of LDL is the product of these two factors. Accord- ingly, any variation in the plasma LDL concen- tration directly and immediately affects the quantity of LDL delivered to the intima. Even in arteries without atherosclerosis, the arterial wall permeability to LDL varies between individuals by a factor of at least 10 [7] implying that individ- uals with a low plasma LDL concentration may experience a relatively high flux of LDL into the arterial wall if the LDL permeability of the arte- rial wall is high. This emphasizes the need for further understanding of the determinants of the lipoprotein permeability of the arterial wall in vivo.

3. Quantification of the arterial wall permeability to LDL in vivo

The LDL permeability of the luminal surface of the arterial wall has been measured in vivo on the basis of accumulation of radioactivity in the arte- rial wall after an intravenous injection of labeled LDL. Most studies have used LDL labeled in the protein moiety with 1251 or 13’1 (Table 1). Others have used LDL labeled with [3H]leucine [8], E3H]- or [14C]cholesteryl ester [7,8], fluorescent com- pounds [9,10], or with [‘251]tyramine cellobiose [6,111.

A large number of studies has estimated LDL permeability by dividing total radioactivity in the arterial wall by the mean plasma radioactivity concentration during the experimental period and by the length of the exposure time [4-8,l l-191. Other studies have used mathematical analysis of transmural radioactivity concentration profiles of [1251]LDL to estimate the LDL permeability of the luminal surface of the arterial wall, and addition- ally to describe transport characteristic of LDL within the arterial media [20-221; the concentra- tion of [1251]LDL in the arterial tissue has been determined after microsectioning the arterial wall parallel to the intimal surface followed by gamma-counting of the tissue slices [22] or by quantitative autoradiography of histological cross sections of the arterial wall [20,21]. Studies of this type indicate that LDL primarily enters the in- tima-inner media from the luminal side and that structures in the intima, presumably both the endothelium and the internal elastic lamina [23- 251, provide significant barriers towards the entry of plasma macromolecules into the inner media. More recently, quantitative autoradiography of histological preparations of en face preparations of the endothelium has been used to measure the LDL permeability in areas corresponding to a few endothelial cells [26,27].

Measurement of the LDL permeability is com- plicated by a number of errors from different sources, including plasma contamination, loss of labeled LDL from the intima during the experi- mental period, and inability of the labeled prepa- ration to mimic the metabolism of the native LDL particle. In studies using LDL labeled in the lipid moiety, exchange of labeled cholesteryl esters or labeled free cholesterol from LDL to other lipo- proteins should be taken into account [7,8].

With an LDL permeability of about 30 nl/cm2/ h in the normal arterial wall (Table l), plasma contamination of the vessel is a significant source of radioactivity in the arterial wall specimen, espe- cially after a short exposure period and at arterial sites with a low permeability (see discussion on regional variation in LDL permeability below): following thorough rinsing of the arterial wall with saline, plasma contamination has been esti- mated to be 5- 10 nl/cm2 using labeled red blood

Tabl

e I

Exam

ples

of

es

timat

es

in v

ivo

of

the

LDL

perm

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uman

ar

terie

s w

ith

mild

at

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scle

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rimen

tal

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Spec

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Ref

eren

ce

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ry

Part

of

Trac

er

inve

stig

ated

ar

teria

l W

all

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mol

ecul

e

Hum

un

[12]

i7]

Aorta

, re

nal

and

fem

oral

ar

terie

s,

<75%

pl

aque

As

txm

ding

ao

rta,

< 10

%

plaq

ue

Mon

key

[13]

En

tire

aorta

~321

En

tire

aorta

PI

Des

cend

ing

aorta

Rab

bit

[15]

Th

or&c

ao

rta

[171

Th

orac

ic

aorta

1141

["I

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acic

ao

rta

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acic

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acic

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rta

~291

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orac

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Th

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Intim

a +

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r m

edia

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ner

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edia

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acco

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loss

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edia

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+ 9'

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lity

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entra

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dete

rmin

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tora

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raph

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30 m

in

or

( + )

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lh

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ose-

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[‘=I]L

DL

6 h

[“‘I]L

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-

[“‘I]L

DL

10 m

in

--

33".'

30'

72

The

LDL

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seria

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rial

wal

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th

e en

doth

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m

wer

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mm

a co

unte

d

w

P

Tabl

e 1

(con

tinue

d)

Spec

ies R

efer

ence

Arte

ry

inve

stig

ated

Pa

rt of

Tr

acer

mol

ecul

e Exp

osur

e Pl

asm

a LD

L pe

rmea

bilit

y N

otes

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teria

l wal

l Pe

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cont

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l/crr

?/h)

an

alyz

ed

corre

cted

m

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on [6

]

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&c a

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tima

[=‘I]

LDL

10m

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- 68

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usi

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athe

mat

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the

endo

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o qu

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all

radi

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di

vide

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the

mea

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able

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]. “C

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f [1

3].

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cont

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n w

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stim

ated

to

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ce a

n av

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% o

vere

stim

atio

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sDat

a w

ere

calc

ulat

ed a

ssum

ing

a su

rface

are

a of

28

cm*/g

wet

wt.

of t

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tima

+ w

hole

med

ia (

Tabl

e 3

of [

ll]).

hRea

d fro

m F

igur

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of [

ll].

‘Cal

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dat

a in

Tab

les

1 an

d 2

of [

82],

assu

min

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dry

defa

tted

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ght

equa

l to

25%

of

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ght

and

a su

rface

are

a of

40

c&/g

w

et w

t. of

the

in

tima

+ in

ner

med

ia.

$ G

L.B. Nielsen / Atherosclerosis 123 (1996) I 15 5

cells [14,28] or labeled LDL [5,17], although some studies using iodinated LDL or cholesteryl ester labeled plasma lipoproteins have found a plasma- contamination of about 30 nl/cm2 [15,29]. In a study by Tompkins et al. [30], the amount of labeled LDL in the arterial wall of monkeys after 30 min exposure was higher when determined by gamma counting compared to autoradiography. This led the authors to suggest that plasma contamination may be of less importance when arterial wall radioactivity is quantified by autoradiography.

The commonly used approach of dividing the radioactivity in the arterial wall by the mean plasma concentration of labeled LDL and by the length of the exposure period in order to estimate the LDL permeability cannot distinguish between luminal and abluminal entry of LDL. Nevertheless, up to 4 h after an intravenous injection of iodinated LDL in normal rabbits, transmural concentration profiles of TCA-precipitable radioactivity in the aorta had step gradients near the intimal surface, moderate gradients near the medial-adventitial border, and were relatively flat in the middle of the media [22]. Thus during exposure periods < 4 h, radioactivity in the intima-inner media primarily reflects luminal entry of LDL.

The above mentioned approach for estimating arterial wall permeability to LDL furthermore relies on the assumption that the arterial tissue acts as a sink during the experimental period [31], i.e. that loss of labeled LDL from the arterial tissue by efflux and/or degradation of labeled LDL is small compared to the influx of labeled LDL. This ‘sink assumption’ has been validated by showing that the arterial wall radioactivity content divided by the mean plasma radioactivity concentration showed a nearly linear increase after 3 h exposure to iodi- nated LDL in the normal aortic arch and after 1 h exposure in the normal thoracic aorta of rabbits [14,17,29]. Others have used the accumulation of labeled LDL in the arterial wall after different periods of exposure to correct for loss of labeled LDL from the intima-inner media in the calculation of LDL permeability [32-351.

Small disintegration products of labeled LDL may enter the arterial wall faster than the intact LDL particle simply due to their smaller size [7,8,35-37] and consequently may produce an

overestimation of the LDL permeability. Such disintegration products may be formed either dur- ing isolation and labeling of LDL, or by cellular processing of labeled LDL in vivo. For instance cellular processing of iodinated LDL, which is often used in permeability studies, results in the presence of non-protein bound radioactivity in plasma, which may rapidly equilibrate with the extracelluar fluid in arterial tissue [38]. The interfer- ence from accumulation of non-protein bound radioactivity in the arterial tissue can be eliminated by precipitation of proteins in the arterial tissue, e.g. with trichloroacetic acid or by fixation of the tissue, and using only precipitable radioactivity in the calculations. The relative contribution of non- protein bound radioactivity to the total amount of radioactivity in the arterial wall depends on the amount of non-TCA precipitable radioactivity that is generated after intravenous injection of iodinated LDL and on the magnitude of the influx of LDL. In normal rabbits on average 47% of the radioac- tivity in aortic intima-inner media precipitated with proteins 3 h after an intravenous injection of iodinated LDL [ 171, whilst in cholesterol-fed rab- bits with atherosclerotic lesions in the aorta, > 90% of the radioactivity in aortic intima-inner media precipitated with proteins [ 191. Oxidative modifica- tions of LDL during isolation and labeling may increase its uptake by arterial wall cells [lo, 131, which could also lead to an additional overestima- tion of the LDL permeability.

4. LDL permeability of the normal arterial wall

Table 1 lists a number of examples of in vivo estimates of LDL permeability of a normal arte- rial wall. The estimates vary between zz 5 and z 100 nl/cm2/h (Table 1). This is much less than an estimate of the water flux across the normal rabbit arterial wall of about 11000 nl/cm2/h at 75 mmHg [39], suggesting that the luminal layer of the arterial wall forms a very tight barrier towards the entry of the = 25-nm sized LDL particle. The considerable variation between studies in esti- mates of LDL permeability of normal arterial wall intima may at least in part be related to the methodological issues discussed in the previous

6 L.B. Nielsen / Atherosclerosis 123 (1996) I- I5

section, but also the large variations in permeabil- ity to LDL between different arterial sites [6,11,17,21,40-421 and between individuals may complicate the comparison of results from different studies.

5. Mechanism for transfer of LDL into the arterial wall

LDL enters the arterial wall as intact particles, although free cholesterol and phospholipid proba- bly exchange between endothelial cells and the surface layer of the LDL particle [37]. In choles- terol-fed rabbits, the flux of LDL into the aorta was the same for LDL labeled in the protein moiety with [3H]leucine and for LDL labeled in the cholesteryl ester moiety with [14C]cholesterol [S]. The presence of newly entered LDL particles in the aortic intima after an intravenous bolus infusion of LDL also’has been demonstrated directly [43] and LDL-like particles can be isolated from the human arterial intima [44].

Several lines of evidence suggest that transfer of LDL into the intima is independent of LDL-recep- tors on the endothelial cells. The LDL permeability of the arterial wall in rabbits was not affected by methylation of the apolipoprotein B in LDL, which abolishes its recognition by the LDL recep- tor [14]. Saturation of LDL receptors by an acute increase in the plasma LDL concentration prior to the determination of LDL permeability, either by an intravenous injection of LDL [22] or by choles- terol-feeding for 3 days [14], did not affect the LDL-permeability. Finally, the relative rates by which LDL and other plasma macromolecules such as albumin and high density lipoproteins are transferred into the arterial wall depend on macro- molecular size, suggesting a passive sieving of LDL and other plasma macromolecules across the en- dothelium [7,8,19,35-371.

Two different pathways for the transfer of LDL across the endothelium have been proposed: (1) ferrying of LDL in vesicular bodies across the endothelial cell (transcytosis) [45-471 or (2) sieving through porous pathways between or through the endothelial cells [48]:

In ultrastructural studies of the aortic endothe-

lium after a bolus injection of labeled LDL [46] and p-VLDL particles [47] into rats and rabbits, re- spectively, labeled particles were found predomi- nantly in vesicular structures within the endothelial cells and later appeared underneath the endothelial cells. This suggests that LDL is transported mainly in vesicles through the endothelial cells [45]. Anal- ysis of ultrathin serial sections of frog muscle endothelial cells however has challenged the exis- tence of vesicular structures in capillary endothelial cells: such studies indicate that the apparent vesi- cles in endothelial cells primarily represent invagi- nations of the endothelial cell membrane [49]. Whether this also is the case in arterial endothe- lium is unknown.

In contrast to the transcytosis theory, mathe- matical considerations support the idea that pas- sage of LDL via pores through or between endothelial cells can account for the bulk transfer of LDL into the intima, even if such pores occupy less than 10 - ’ of the surface area [48] and thus are unlikely to be detected by conventional morpho- logical examinations. It has been suggested that areas of endothelial cell division [9,26] and death [50] may provide porous pathways for passage of LDL into the intima. A recent ultrastructural study detected open junctions of a width of 25-300 nm between endothelial cells of the rat aorta [51].

6. Increased arterial wall permeability to LDL and initiation of atherosclerosis

The focal nature of plaque formation is a strik- ing feature of atherosclerosis (reviewed in [52]). A number of studies using different techniques favour the idea that a high permeability to LDL at certain arterial sites is closely related to the propensity for development of atherosclerosis at such sites.

Upon intravenous injection, labeled plasma proteins and labeled cholesterol accumulate prefer- entially in atherosclerosis susceptible segments of aorta in pigs and dogs [40,53,54]. More recently, a strong correlation between the LDL permeability of a certain aortic segment in normal rabbits and pigs and the severity of atherosclerosis in the same segments after cholesterol-feeding has been demonstrated [17,41] (Fig. 1). Although one

L.B. Nielsen / Atherosclerosis 123 (1996) I 15 I

study in rabbits did not find a difference in perme- ability between the susceptible arch and the less susceptible thoracic aorta [1 11, a later study from the same laboratory did [55]. In addition to the variation in permeability along the length of the aorta, studies in rabbits support the idea that the permeability to LDL is increased at atherosclero- sis susceptible branch sites of the aorta compared to adjacent atherosclerosis resistant non-branch sites [11,26,42]. In contrast, no association be- tween the LDL permeability of a given aortic site and the subsequent development of atherosclero- sis at that site was found in the pigeon [6].

Using horseradish peroxidase, Sterner-man et al. detected foci in the normal rabbit aorta with a luminal surface area of about 0.1 mm2 which had an LDL permeability up to 47 times greater than surrounding regions [56]. The number of high permeability foci per cm2 ranged from 150 in the atherosclerosis susceptible aortic arch to 15 in the

“f- b 20 40

Aortbc permeability to LDL hllcm’lhr)

Fig. 1. The LDL permeability is larger in atherosclerosis prone compared to atherosclerosis resistant aortic segments of the rabbit. The ligure depicts the association between aortic LDL permeability in a given aortic segment of normal rabbits on the x-axis and cholesterol content in that segment after 3 months of cholesterol-feeding of other rabbits on the y-axis. Permeability and cholesterol accumulation was determined in 12 consecutive segments of the aorta. Each point represents the mean values in a given aorric segment for 11 rabbits. Permeability to LDL was determined from the plasma con- tamination corrected accumulation of iodinated LDL in the aortic intima-inner media after 3 h exposure [17]. Reproduced with permission from the American Heart Association.

more resistant descending aorta and were more frequent around ostia of aortic branches [57]. The existence of foci with elevated LDL permeability are supported by other studies in rats, rabbits and monkeys [9,21,26]. A detailed analysis of the dis- tribution of sites with a high LDL permeability around intercostal and coeliac arteries of the rab- bit aorta, revealed that highly permeable sites occurred most frequently at the distal and lateral edges of the orifices [26], which are the most atherosclerosis susceptible sites; the larger fre- quency of high permeability sites could account for the increase in LDL permeability around orifices compared to neighbouring sites [26].

The use of histochemical and autoradiographic methods have allowed quantitative estimates of macromolecular permeability to be related to his- tological structures [20,21,23,58]. Such studies in- dicate the existence of foci with a markedly increased permeability which are covered by an apparently intact endothelium [30,56,57]. By his- tological examinations of en face preparation of normal rat aortic endothelium, Lin et al. [9] de- tected focal leakage of fluorescence labeled LDL and found that 45% of all leakage foci were associated with endothelial cell mitosis. On en face autoradiography of the endothelium of nor- mal rabbit aorta after in vivo exposure to [‘=I]LDL, 70-90% of mitotic endothelial cells showed an increased permeability to LDL [26,27]. However, only 13% of the sites with an increased permeability, were associated with mitotic cells [26]. Cell death [50] or wounding of endothelial cell membranes [59] may also be involved in fo- cally increased LDL permeability. Intercellular gaps are more frequent in the branch compared to the non-branch regions of the rat aorta [51]. The mechanism for cell damage at certain arterial sites may be related to variations in shear stress at branch sites and disturbed flux behind the aortic valves [60,61]; high shear stress has been shown to increase the arterial wall permeability to labeled albumin in vivo [62].

Although there is a growing consensus that a high LDL permeability is located at sites with a high risk for development of atherosclerosis, it can be debated whether this association reflects a causal relationship [63]. Mechanical [64,65] or im-

8 L.B. Nielsen / Atherosclerosis 123 (1996) l-15

munological injury [16] increases the arterial wall permeability and accelerates subsequent develop- ment of experimental atherosclerosis; these find- ings favour a causal relationship. On the other hand, the LDL permeability of veins, aortic valves, and pulmonary arteries is markedly higher than that in the aorta [21,31,66] although atherosclerosis rarely develops in veins, valves or the pulmonary artery. The latter observations em- phasize that although an increased arterial wall permeability may increase the risk of atherosclero- sis other factors such as lipoprotein retention may also play a role. Studies in rabbits by Schwenke and Carew [11,67,68] suggest a preferential reten- tion of LDL after its entry into the arterial wall in the atherosclerosis susceptible aortic arch and aortic branch sites compared with the more atherosclerosis resistant sites of the aorta [l l] and that increased retention of LDL rather than in- creased permeability preceded the development of atherosclerosis in cholesterol-fed rabbits. More- over, microscopic foci with an elevated LDL per- meability may also be characterized by an increased binding of LDL to subendothelial com- ponents [27,57]. Therefore, not only an increased influx but also an increased retention of LDL may contribute to focal accumulation of LDL and early development of atherosclerosis.

7. Increased LDL permeability in atherosclerotic lesions

It is firmly established that once atherosclerosis develops, the LDL permeability of the vessel wall increases markedly [5-7,12,13,32], e.g. the LDL permeability was z 6 times higher in atheroscle- rotic aortas of monkeys compared to non- atherosclerotic monkeys [13]. Such increased permeability in atherosclerotic plaques may en- hance the focal development of lesions, however, the mechanism is unknown. Gross endothelial loss probably does not occur in atherosclerosis [l], therefore, dysfunction of the intact endothelial layer [69] provides a more likely explanation for the increased arterial wall permeability in atherosclerosis. Endothelial cells overlying atherosclerotic lesions have an altered morphol-

ogy [70] compared to endothelial cells of normal arteries. Endothelium dependent relaxation is im- paired in atherosclerotic arteries, possibly due to a defective synthesis of nitric oxide [71]. Interest- ingly, inhibition of nitric oxide synthesis by nitro- L-arginine methylester (L-NAME) promoted reperfusion-induced vascular leakage of albumin in rats [72] and accelerated the development of atherosclerosis in cholesterol-fed rabbits [73]. However, it remains to be investigated how an impaired nitric oxide system may affect the trans- fer of LDL into the arterial wall in vivo.

Enhanced migration of monocytes into atherosclerotic lesions may also augment the LDL permeability at the lesion sites, e.g. because of coincident leakage of lipoproteins at sites of monocyte penetration of the endothelium. The migration of monocytes across cultured endothe- lial layers has been coupled to an increased transendothelial transport of LDL [74,75] and recently it was demonstrated that reperfusion-in- duced leakage of albumin from post-capillary venules can be inhibited by monoclonal antibod- ies to leucocyte adhesion molecules CD18, CD1 lb, ICAM- and L-selectin [76].

8. Risk factors for atherosclerosis and arterial wall permeability to LDL

Hypercholesterolemia, hypertension, smoking, familial predisposition to ischemic heart disease and male sex are well established risk-factors for atherosclerosis [3]. These risk factors potentially could affect the interaction of LDL with the arte- rial wall.

The flux of LDL into the arterial wall increases in proportion to the plasma LDL cholesterol level as discussed above. Hypercholesterolemia per se has been proposed to increase the arterial wall permeability without any apparent loss of the endothelial integrity: in endothelial cell cultures, incubation with LDL and B-VLDL [77,78] in- creased the endothelial permeability to LDL. Cholesterol-feeding of rabbits for 2 weeks in- creased the frequency of horse radish peroxidase leaking spots in the lesion free aortic endothelium [79]. Similarly, Menzoian found an increased

L.B. Nielsen / Atherosclerosis 123 (1996) I- 15

transport of albumin across the rabbit carotid arterial wall after 1 week of hypercholesterolemia [80]. The mechanism for the potential effect of hypercholesterolemia on permeability to smaller particles such as albumin and horse radish perox- idase may be related to increased endocytotic activity and formation of stress fibres in endothe- lial cells [81], or to a decrease in basement mem- brane content of heparan sulphate proteoglycans [77]. Schwenke and Carew. however, did not find any evidence for an increased aortic permeability to LDL in rabbits after 4, 8, or 16 days of cholesterol-feeding [l l] and hypercholesterolemia in pigeons for up to 10 months did not increase the aortic wall permeability to LDL in the ab- sence of atherosclerosis [6]. Studies from the au- thors laboratory have also not been able to detect an increased LDL permeability of the rabbit aorta after 1 week of cholesterol-feeding (unpublished data). The notion that hypercholesterolemia per se increases the arterial wall LDL permeability in vivo therefore has little support. The divergent effects of hypercholesterolemia on arterial wall permeability to albumin and LDL emphasizes the notion that arterial wall permeability to small molecules not necessarily reflects permeability to LDL, as suggested by Schwenke and Zilversmit P51.

Transfer of LDL into the arterial wall pre- sumably is increased by high blood pressure. Af- ter 6 h exposure in vivo, the accumulation of [125J]LDL in the aortic intima-inner media of rab- bits that had been hypertensive for 1 month and in similar rabbits where blood pressure had been acutely normalized was increased compared to normotensive rabbits [82]. Similarly, hypertension for 1 week in rats increased the albumin perme- ability of the abdominal aorta even when blood pressure was acutely normalized [83]. These re- sults may suggest that an increased flux of LDL into the intima during hypertension is mediated in large part by increased macromolecular perme- ability of the arterial wall. However, the increased accumulation of iodinated LDL after 6 h expo- sure may have resulted from an increased volume of distribution for labeled LDL and as discussed above, results obtained with albumin cannot auto- matically be extrapolated to LDL. Other studies

Angiotensm II Saline Meal BP (mmHd -

107f3 82k7 82f4

Fig. 2. The flux of LDL into the aortic arch intima-inner media of rabbits is increased by acute elevation of blood pressure. The figure depicts normalized flux of LDL into aortic intima-inner media in conscious rabbits during continuous infusion of angiotensin II (1.4 /cg/kg/min) (left and middle bars) or saline (right bar). Angiotensin II infusion initially increased blood pressure, but this effect vanished after 1-2 h where blood pressure returned to baseline values, despite continuous infusion of angiotensin II. *P i 0.05 compared with angiotensin II infused rabbits with high blood pressure. Normalized influx of LDL was determined from the plasma contamination corrected accumulation of iodinated LDL in the aortic intima-inner media after 1 h exposure [15]. Repro- duced with permission from the American Heart Association.

support a direct effect of blood pressure: the flux of LDL into the rabbit aorta ex vivo was larger at 160 mmHg compared with 70 mmHg [84], and Fry et al. found a pressure dependant uptake of LDL by the de-endothelialized, but not by intact intima in isolated aortic segments from pigs [85]. In vivo, continuous infusion of angiotensin II and noradrenaline concomitantly increased blood pressure and flux of LDL into the rabbit aorta [15]. However, after 2 h blood pressure and the flux of LDL into the arterial wall returned to normal values, despite continuous infusion of an- giotensin II (Fig. 2). Thus, an acute increase in blood pressure may augment transfer of LDL into the intima, e.g. by pressure driven convection. Such a mechanism has also gained experimental support from the pressure dependency of LDL transport across microvessel endothelium [86,87].

The mechanisms by which blood pressure affects transport of macromolecules into the in- tima is unclear. Different types of hypertension affect the morphology of the endothelium differ- ently [88,89]. Renal hypertension in rats induced an increased vesicular transport of ferritin across

10 L.B. Nielsen 1 Atherosclerosis 123 (1996) l-15

Smoking Genetic predisposition Atherosclerosis Shear stress Small size of LDL

A--

t High LDL-permeability

High plasma LDL concentration

t

High blood pressure

Increased flux of LDL into the arterial wall

t Accelerated development of atherosclerosis

Fig. 3. Hypothetical schema showing determinants of increased flux of LDL from plasma into the arterial wall, and possibly subsequent accelerated development of atherosclerosis. A high concentration of LDL in plasma as well as hypertension may directly augment the arterial wall influx of LDL. The latter may occur by increased convective transfer of LDL into the arterial wall without any change in arterial wall permeability (lower arrow); hypertension may in addition increase arterial wall LDL permeability (upper arrow). Smoking, genetic predisposition, atherosclerosis, shear stress, and small size of LDL may also increase the LDL permeability of the arterial wall.

the aortic endothelium [90], whereas a later study found an increased turnover of endothelial cells and attendant enhancement of the Evans Blue dye labeled albumin permeability in chronic hyperten- sive rats [91].

In addition to its pressor effect, some studies suggest that sympathetic activation increases the arterial wall permeability to LDL by a non-pres- sor mechanism: infusion of adrenalin directly into the carotid artery of rabbits increased the uptake of labeled LDL after 1 h exposure in vivo [92] and sympathetic activation by chloralose increased the number of dying or dead endothelial cells in the aorta of rabbits [93]. Another study, however, could not demonstrate an effect of sympathetic activation by chloralose on the arterial wall lipo- protein permeability in normal rabbits [94]. The latter observation suggests that endothelial cell death not necessarily results in an increased lipo- protein permeability.

Angiotensin II, another potent pressor molecule, has also been suggested to increase the albumin permeability of the rabbit aorta in vivo via a non-pressor mechanism [95]; angiotensin II has been shown to induce an opening of intercel-

lular junctions in the endothelium [96]. Regardless of these findings, a later study in rabbits could not support a major direct effect of angiotensin II on the LDL permeability of the aortic wall [15]. These divergent data may result from different experimental conditions or may reflect that inter- ventions that influence arterial permeability to albumin not inevitably influence arterial wall per- meability to LDL. Endotoxins may also increase the macromolecular permeability of the arterial wall [97], but the importance of this finding for atherosclerosis is unclear.

Administration of nicotine to rats increased the number of dead or dying endothelial cells in the aorta and also increased transendothelial leakage of albumin [98]. Whether nicotine increases arte- rial wall permeability to LDL and in this way may contribute to accelerated formation of atherosclerosis in smokers is unknown.

The idea that a high LDL permeability may be inherited has been supported by the finding of a positive association of the arterial wall permeabil- ity to plasma lipoproteins between rabbit siblings [94]. Another study, however, could not confirm such an association in ovariectomized female rab- bits [18].

L.B. Nielsen / Atherosclerosis 123 (1996) 1- 15 11

The increased prevalence of atherosclerotic dis- ease in men compared with women may relate to an anti-atherogenic effect of estrogen. Estrogen is capable of restoring a defective endothelial depen- dant relaxation of atherosclerotic coronary arter- ies [99], indicating a direct effect of estrogen on endothelial cell function. Haarbo et al., however, found no effect of estrogen therapy on the LDL permeability of the normal rabbit aorta [18], whereas Wagner et al. found that estrogen and progesterone replacement therapy reduced the combined accumulation of LDL and products of LDL degradation in arteries of ovariectomized monkeys [ 1 OO]. These two studies together suggest that the beneficial effect of estrogen is related to a decreased degradation of LDL in the arterial in- tima rather than to an effect on endothelial per- meability. This idea is supported by the findings of reduced hydrolysis of newly entered cholesteryl esters [loll and of decreased degradation of LDL in the arterial wall of monkeys after treatment with oral contraceptives [ 1021.

9. Chemical modification of the LDL particle affects its transfer into the arterial wall

In addition to endothelial factors, the composi- tion of the LDL-particle affects its transfer into the intima under certain conditions. Acetylation of [‘251]LDL increased its transfer into the aortic wall in monk.eys 20 min after an intravenous injection [ 131. Incubation of fluorescence labeled LDL with leucocytes prior to intravenous injec- tion also increased the accumulation of fluores- cent material in aortic endothelial cells of rats in vivo [lo]. Whether chemical modification of LDL in vivo, e.g. by oxidation and glycosylation, affects its transfer into the arterial wall in humans is unknown.

10. Concluding remarks

The present evidence favours the idea that arte- rial wall permeability to LDL is increased at atherosclerosis prone sites in the aorta of experi- mental animals. This suggests that a high arterial

wall permeability to LDL, as well as a high concentration of LDL in plasma, may directly cause accelerated development of atherosclerosis. However, as discussed above, increased retention of LDL may also contribute to development of early atherosclerosis.

Fig. 3 summarizes some possible determinants of an increased flux of LDL into the arterial wall. High plasma LDL concentration, high blood pressure and smoking are all well established risk factors for atherosclerosis. The schema in Fig. 3 outlines the hypothesis that at least part of the atherogenic effect of these risk factors is mediated via increased flux of LDL into the arterial wall. The schema also emphasises the possibility of reducing atherosclerosis risk at the level of the arterial wall, e.g. by improving the barrier func- tion towards entry of LDL. Such a strategy how- ever awaits identification of agents which directly modulate the LDL permeability of the arterial wall.

Acknowledgements

Thanks to Drs Barge G. Nordestgaard and Steen Stender for constructive criticism of the present paper.

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