Permeability patterns in pig aorta

Atherosclerosis Elsevier Publishing

PERMEABILITY I;. H. KLYNSTRA

Company,

Amsterdam

PATTERNS

-

IN PIG AORTA

AND C. J. F. BOTTCHER

.4 therosclerosis Research Unit of the Organization 1Jniversity of Leyden, Leyden (The Netherlands) (Received

November

451

Printed in The Netherlands

for Health

Research

TNO,

Gaubius

Institute

of the

25th, 1969)

SUMMARY

The preferred localizations of fatty streaks and spots in the thoracic pig aorta are shown to have enhanced permeability. The fact that these places are more readily attacked by mucolytic and proteolytic enzymes, which results in a further increase in permeability, indicates that enzymatic attack is one of the factors involved in the development of atherosclerosis.

Key words: Atherosclerosis Proteolytic

- Mucolytic

enzymes

enzymes - Vascular

- Pig aorta - Predilection

places -

permeability

INTRODUCTIOK

Much of the current research on atherogenesis is based on the supposition that an excess of some component of the diet leads to too much of something in the blood, as in hypercholesterolaemia, hyperlipidaemia, and primary hyperlipoproteinaemia, and that this in turn leads to too much of something in the vessel wall. A direct infiltration process from the lumen into the arterial wall would easily explain the last step in this sequence of events. One of the most striking features of atherosclerosis is the patchy distribution of the lesions: different parts of the vascular tree, as well as different segments of a given artery, show wide differences in their degree of involvement. In the involved area the lesions show a characteristic pattern, commonly consisting of many small discrete spots and streaks in a longitudinal arrangementr. The patchy distribution of the early lesions in both man and other species has been seen as a phenomenon that is difficult to reconcile with the filtration theoryz, although attempts have been made to correlate the distribution pattern of the lesions with such haemodynamic factors as turbulence and flow patternrla. The work reported in this paper contributes to a solution of this controversial point. Atherosclerosis,

1970, 11: 451-462

452

F. B. KLYNSTRA,

According aortas

to our previously

were incubated

solutions

with

were replaced

published

mucolytic

by diluted

surface

showed

increased

surface

closely

resembled

of experiments

and proteolytic

Indian

permeability. that

results

C. J. F. BiiTTCHER

enzymes

ink, some well-defined Surprisingly,

of the fatty

streaks

pig

the enzyme

areas of the inner

the pattern and

in which

before

of the blackened

spots

in atherosclerotic

aortas4. The present inner

surface

communication

of pig aortas

deals with studies

in response

that

zymes.

It will be shown

related

to the topography

of local differences

young,

apparently

aortas.

normal,

on these local differences

to the permeability-increasing

the patchy

distribution

of the early lesions

in permeability

The assumption that the three-dimensional networks are complexes of chondroitin sulphates A and C and proteins to investigate MATERIALS

the action

of mucolytic

and proteolytic

action

already

regulating determined

of the of en-

is closely existing

in

permeability our decision

enzyme+7.

AND METHODS

Fresh pig aortas were obtained from the local slaughterhouse, transported on ice, and used within 2 h after the slaughtering of the animal. Two age-groups were chosen for these experiments, one including young pigs of about 6 months and the other animals of 2 years and older, previously kept as breeding-stock. The aortas of the young pigs were free of visible ones regularly

showed fatty

The experiments

atherosclerotic

streaks

were performed

distal end and the lateral

arteries

lesions,

enzyme-containing buffer solutions, bated at 37°C. In some experiments

plaques.

with fresh aortic tubes about

of this tube were ligated,

tested for leakage. The water-tight

whereas those of the older

and spots, and sometimes

sacks obtained

25 cm long. The

and the preparation

was

in this way were filled with pure

hung in physiological pressure was applied

or

salt solution, and incuby inserting a tight-fitting

glass tube into the open end of the aortic preparation. The glass tube was connected an airpump, valves, and a manometer. This method offers the great advantage

to of

protecting the endothelium against mechanical damage. The buffers consisted of Tyrode’s fluids and acetic acid-sodium acetate (pH 6.0, 0.1 M). The enzymes were testicular

hyaluronidase

(NBC, 300 USP/mg

and Serva, 500 USP/mg),

/I-glucuronidase

(Serva, 400 Fishman U./mg), trypsin (NBC, 2 x tryst., 8000 S.T.U.), collagenase (NBC, ammonium sulphate fractionated 2 x), elastase (Sigma, 33 U./mg), and plasmin (NBC, 1 mg/ml liquefies in 1 h a clot from 1 ml whole blood). The effect of the enzymes on the permeability of the inner surface of the aorta was observed by replacing the incubation medium inside the aorta preparation by a 1 : 10 dilution of Indian ink (Pelikan No, 17, black, particle size 0.15-0.8 p) at room temperature. After 18 h of exposure to the Indian ink, the aorta was opened longitudinally and its inner surface photographed. The depth of penetration was observed microscopically in cross-sections. Routine histological staining procedures were used on formol-frozen, 10 p thick sections. Unstained material was compared with sections Atherosczerosis, 1970, 11: 451-462

PERMEABILITY

PATTERNS

453

IN PIG AORTA

stained

for elastic

method

and with others stained

fibres according

In some experiments

to Lawson’s

modified

with haematoxylin

the incubation

blue (molwt. 960) or 1% human stead of Indian ink.

was performed

haemoglobin

Weigert

resorcin-fuchsin

and eosin. with aqueous

(mol.wt.

0.1% trypan

68,000) in Tyrode’s

fluid in-

RESULTS

Permeability

of the untreated

To obtain carried

aorta

information

about

out in fresh untreated

material

the situation

aortas.

Under

not exposed to enzyme-containing

with Indian

ink, trypan

(1) The untreated

in viva, 4 series of experiments

“untreated

aortas”

is understood

or pure buffer solutions

these results

blue or haemoglobin. inner surface of thoracic

aortas was found to be impermeable Application of (18 1~) did not

appreciably.

(2) Incubation under a pressure

fresh

before incubation

and the abdominal part only slightly permeable to Indian ink particles. a pressure of 5 cm Hg to the Indian ink solution during incubation change

were

with

a 0.1%

trypan

of 5 cm Hg, resulted

blue (molwt.

in intensive

960) solution

homogeneous

during

staining

18 11,

of the inner

surface. After cutting pattern

down the incubation

was obtained

(Fig.

conditions.

The individual

commonly

they

time to as little

1). More than variation

were composed

100 aortas

in the resulting

of numerous

as 2-3 min a very distinct were examined

patterns

distinct

longitudinal,

sinuous lines. Their distribution resembled the characteristic sudanophilic streaks and spots in atherosclerotic aortas. In an additional pressure

series (4 aortas),

oxygenated

patterns

these

large, but

more

localization

buffer solutions

was raised to 11 cm Hg. No effect on the resulting

under

was rather

or less of the

were used and the was observed,

nor

any effect on their distribution. (3) Incubation 17 h, under

with haemoglobin

a pressure

(mol.wt. 68,000) instead

of 5 cm Hg, led to weak reddening

intensity of the staining, however, was not uniform, darker red could be seen. These patterns closely resembled those described

of trypan

of the inner

distinct

blue, during surface.

longitudinal

above. They obviously

The

areas of repre-

sent areas where the rate of penetration of these dyes is larger than in the surrounding inner surface layers. Substances of small molecular size, such as trypan blue produce these patterns within 3 min, but stained the inner surface completely in about 30 min. The penetration rate of the relative large haemoglobin molecules is much lower, for here the characteristic pattern was still present after 17 h of incubation. To particles as large as Indian ink the inner surface proved to be impermeable, independent of the time of incubation. (4) Sudanophilic (fatty) streaks and spots were often found in the aortas of old breeding-stock animals (Fig. 2), rendering them very useful to acquire information on Atherosclerosis,

1970, 11: 451-462

F. B. KLYNSTRA,

Fig. 1. Inner surface of thoracic parts of frer for 3 min with trypan blue, under a press1

Atherosclerosis, 1970, 11: 45 l-462

P’-eparc 3f 5 cm

C. J. F. B6TTCHER

1Pig a01-tas (6 m.onth s old) al%er inc .ubakion %.

PERMEABILITY

PATTERNS

IN PIG AORTA

Fig. 2. Inner surface of thoracic parts of pig aortas from old breeding-stock males, stains:d Sudan IV. showing patterns of the fatty streaks and spots.

.~&~ruscZerosis, 1970, 11: 451-462

F. B. KLYNSTRA,

456

C. J. F. BiiTTCHER

Fig. 3. Inner surface of the thoracic aorta of an old pig (male). A : after incubation for 3 min with trypan blue under a pressure of 5 cm Hg; B: the same aorta after additional staining ti ith CSudan IV.

Atherosclerosis.

1970, 11: 451-462

PERMEABILITY

PATTERNS IN PIG AORTA

457

Fig. 4. Inner surface of thoracic pig aortas (6 months) incubated with enzyme-cant .aining 1suffer solutions followed by Indian ink (1 : 10; 17 h). A: with trypsin plus hyaluronidas ,e in Tyr .ode’s fluid (resp. 1 mg/ml, 1 mg/ml; 1.5 h). B: with B-glucuronidase (2.5 mg/ml; 5.5 h).

Atherosclerosis,

1970, 11: 451-462

458

F. B. KLYNSTRA,

a possible connection between the areas of enhanced permeability

C. J. F. BiiTTCHER

and these athero-

sclerotic lesions. Therefore, in another series of experiments, untreated thoracic aortas of old pigs were briefly incubated longitudinally

opened and treated

with trypan blue under a pressure of 5 cm Hg; with Sudan IV. Reddish stained sudanophilic

streaks and spots were encountered in 11 of these preparations. they were found to be situated exclusively

Without exception

along the trypan blue stained longitudinal

lines (Fig. 3). Influence

of the bufer

Incubation

solutions

of thoracic aortas for 5 h with Tyrode’s fluid (pH = 8.2), Verona1

buffer (pH = 7.4), phosphate buffer (pH = 7.4), or acetate buffer (pH = 6.0) left the inner surface impermeable

to Indian ink. Incubation

with more acidic buffers, e.g.

acetate buffers at a pH of 5.0 or 4.2, resulted in an increased permeability

of some

areas of the inner wall, as shown by the blackened patterns after treatment Indian ink. These patterns show great individual variability, ly composed of many discrete longitudinal

with

but they too are common-

lines and closely resemble the patterns

described above. The permeability-increasing effect of the acidic buffers may be due to a loosening of the acid mucopolysaccharide (AMPS)-protein linkages, an assumption supported by the fact that appreciable

amounts of aortic acid mucopolysaccharides

could be

detected in these buffer solutions after incubation. Influefice of enzymes The addition of mucolytic or proteolytic enzymes to, the Tyrode’s fluid or acetate buffer (pH = 6.0) resulted in the appearance of blackened areas on the inner surface of the thoracic aortas after treatment with Indian ink. The highly variable patterns of these areas strikingly resembled those found after brief incubation with trypan blue or the Indian ink patterns seen after incubation with acetate buffers at pH = 5.0 and 4.2 (Fig. 4). The resulting trypan blue patterns in aortas from old pigs did not differ from those observed on the inner surface of aortas from young animals. There were marked differences in the permeability-increasing various enzyme preparations

used in these experiments.

effect of the

The most effective

was

elastase, which produced a very distinct pattern in 1 hour at a concentration of 0.25 mg/ml, followed by collagenase (1 h, 0.5 mg/ml), plasmin (1 h, 1 mg/ml), testicular hyaluronidase (Serva, 500 USP/mg: 3 h, 0.4 mg/ml and NBC 300 USP/mg: 5 h, 0.7 mg/ml), trypsin (1 h, 10 mg/ml), and b-glucuronidase (6 h, 10 mg/ml). The depth

of penetration

The examination of about 1500 transverse sections of the aortic tissues after the various incubation experiments proved unequivocally that penetration processes and not adhesiveness underlie the phenomenon under investigation. The fact that the penetrated areas had a more or less homogeneously stained, belt-like appearance suggests that the passage of particles had not been confined to gaps between endothelial cells. Atherosclerosis,

1970, 11: 451-462

PERMEABILITY

459

PATTERNS IN PIG AORTA

Fig. 5. Transverse sections of thoracic pig aortas (6 months of age). 250 x The arrows indicate places showing no permeation (a), permeation of Indian ink particles restricted to endothelium (b), permeation into sub-endothelial layers (c) and the nuclei (n). A : incubated with acetate buffer pH 4.2 (0.1 M) for 5 h followed by Indian ink for 17 h. Stained for elastic fibres according to Lawson’s modified Weigert-resorcin-fuchsin method. B: incubated with hyaluronidase (0.4 me/ml) for 3 h followed by Indian ink for 17 h stained with haematoxylin and esosin.

Fig. 5A shows a transverse section of the intima and adjacent media of an Indian ink-blackened area of the thoracic aorta of a young pig (6 months) after prior incubation with acetate buffer (pH = 4.2). The ink particles are mainly confined to the endothelial layer. At some places the underlying intimal layers are penetrated to a depth of about 10 ,LLVirtually the same picture was seen after prior incubation with hyaluronidase (Fig. 5B) and the other enzymes. The depth of penetration in these experiments usually did not exceed 5-7 ,u, but occasional cases of deeper penetration Atherosclerosis,

1970,

11: 451-462

460

F. B. KLYNSTRA,

were observed,

as shown in Figs. 5A and B. The greatest

into the intimal

layers was found after incubation

The basement trypan

membrane

blue molecules.

aorta incubated

proved

The depth

with trypan

depth

of local penetration

with elastase

not to hinder

of penetration

C. J. F. BGTTCHER

(50 ,u).

the passage of the muchsmaller

into the enzyme-treated

blue for 17 h varied

thoracic

from 50-150 p.

DISCUSSION

Experiments in vivo and in vitro have shown that up to a certain particle size the inner layer of the aorta does not act as an impenetrable barrier. In experiments with et al.9 found

ADAMS

live rabbits,

aortic wall for cholesterol

but not for labelled

enter the wall from the intimal cockerels et

proved

experiments

regional

for trypan

physiological

ic vascular graft material implantation12~1s. We may assume, fulfillment

property

in human

has proven therefore,

inward-outward

direction

the fresh thoracic

The present

thoracic

permeability

pig aortas

that permeabil-

wall, an assumption

the final “biologicalporosity”

functions

upper limit to the size of the particle,

aorta for albu-

These resultssuggest

to be one of the ultimate

that

DUNCAN

in the rate of entrance. prepared

strongly of prosthet-

factors

in successful

of its wall is essential

of the artery.

There

may

aorta of

and phosphoproteinsle.

of the aortic

surgery:

of the biological

of the thoracic

of the inner layer of the canine variations

blue and haemoglobin.

by experience

across the

some of which, however, surface

have shown that the inner layers of freshly

ity is a normal

normal

albumin,

The intimal

to phospholipids

the permeability

and described

are permeable supported

surface.

to be permeable

al.11 demonstrated

min in vitro,

aninward-outwardconcentrationgradient

for the

is, however,

an

above which free passage across the wall in the

is impossible.

This is illustrated

pig aorta for Indian

by the impermeability

of

ink particles.

The vascular permeability in vivo is not constant, and can be altered by biochemical and/or mechanical influences. The fact that various substances, e.g. plasmini4

hyaluronidasers,

permeability-increasing must be controlled AMPS,

together

and angiotensin

16, have been reported

effect, means that passage by one or more regulatory

with

such enzymes

of material

systems.

as hyaluronidase,

to exert

a vascular

across the aortic wall

The hyaluronidase-digestable may represent

such a sys-

terne~7~17. It is assumed that these substances, i.e. hyaluronic acid and chondroitin sulphate A and C, in the form of their complexes with proteins, exert a passagerestricting effect in the aortic wall. This effect is counteracted by the action of mucolytic and proteolytic enzymes, as shown by our experiments. After incubation with these enzymes, the thoracic aortic wall loses its impermeability for Indian ink particles. The increase in permeability is accompanied by a decrease of the AMPS concentration in the tissueI*. Incubation of the thoracic aorta with permeability-increasing enzymes, however, did not render its inner surface uniformly permeable to Indian ink: only some well-defined areas became blackened, while other parts remained unstained. That the increases in permeability are really the result of enzyme activity and not of Atherosclerosis,

1970, 11: 451-462

PERMEABILITY

PATTERNS

other influences,

IN PIG AORTA

e.g. hypoxia,

enzyme-containing

can be readily

and enzyme-free

except the omission

seen from comparison

buffer solutions:

of the enzyme,

shown by this blackened

161

no increase

in permeability

inner surface is composedof

meability-promoting enzymes hyaluronidase the human

leading

atherosclerotic

and b-acetyl sclerosis,

activity

to an increase

intravenously

injected

testicular

hyaluronidase

reported

is an increase found

PLATTlg

increased.

of per-

KASABIJAN~~

of the MPS-hydrolases,

were significantly

lines,

activity

of atherosclerosis

in permeability.

aorta the activities

glucosaminidase

sclerosis-promoting

to note that an increased

factors in the initiation

The pattern

pattern of the sudanoof old breeding stock.

has been found in atherosclerosis.

that one of the pathogenic

being unchanged

occurred.

many discrete longitudinal

sometimes almost confluent, and resembles the distribution philic fatty streaks and spots often encountered in aortas Seen in this light, it is very interesting

of our results with

all parameters

in

that

in

,&glucuronidasc,

In experimental proved

athero-

to exert an athero-

effect in ratGo.

In an earlier publication

we suggested

that the development

of fatty streaks and

spot-like patterns in the enzyme-treated aortas might be due to local differences in the binding strength or density of the ilMPS--protein complexes”. Ourpresent experiments, in which untreated fresh thoracic aortas were incubated with trypan blue and harmoglobin, show the existence of areas having higher accessibility to these molecules than the other parts of the inner surface. with trypan

blue sufficiently,

By cutting

down the duration

we were able to produce

those found after the incubation of enzyme-treated deed indicated the existence of areas of enhanced ently

normal,

thoracic

atherosclerotic

aortas.

staining

with Sudan

aorta

patterns

of the incubation closely

aortas with Indian ink. This inpermeability in the young, appar-

of the pig. These same areas are still found

Incubation

of these older aortas with trypan

IV, showed

that

the patterns

and spots are places of relatively to attack

the passage-restricting permeability

becomes

enhanced

from mucolytic

properties

permeability; and proteolytic

in older,

blue, followed by

of the sudanophilic

spots coincide with the patterns of enhanced permeability (Fig. 3). These findings strongly suggest that the preferred localizations more accessible

resembling

streaks

of fatty streaks

these sites are thought enzymes,

of the tissue of the arterial

and

to be

which diminishes

wall to a level at which

pathological.

ACKKOWLEI>GEMENTS

The pig aortas were generously

provided

by the local slaughterhouses

of Leyden

(Dr. H. Vis) and The Hague (Dr. K. Hofstra). The authors wish to thank Miss L. Verde1 and Mr. W. J. Brokaar for their skilful technical assistance. Dr. E. Boelsma-van Houte contributed valuable help and advice.

.4 therosczerosis,

1970, 11: 151462

F. B. KLYNSTRA,

462

C. J. F. BiiTTCHER

REFERENCES

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Atherosclerosis,

1970, 11: 451-462