Plasma proteins, oxygen transport and atherosclerosis

Atherosclerosis Elsevier Publishing

PLASMA

Company,

PROTEINS,

G. M. CHISOLM,

Amsterdam

OXYGEN

JOHN

TRANSPORT

L. GAINER,

327

- Printed in The Netherlands

AND ATHEROSCLEROSIS

G. E. STOSER

AND JAMES

V. GAINER,

JR.

De@zrtmewtof Chemical

Engineering (G.M.C., J.L.G.), and Department of Materials Science (G.E.S.), University of Vwginia, Charlottesville, I-a., a+ld Division of Neurosurgery (J. V.G.), Schoolof Medicine, West Virginia University, Morgantowz, IV. I’a. (U.S.-4 .) (Received

July 30th, 1971)

SUMMARY

In summary, the formation

we have presented

of atherosclerosis.

following etiology

to support

More importantly,

the hypoxic

though,

theory

for

we have presented

the

for such hypoxia:

(1) Oxygen transport humans probably

is affected by plasma protein concentrations

decreases

tures which results in increased (3) The interior

and in most

with age;

(2) The result of hypoxia structure

at the aortic lining is a degeneration cellular permeability; matter.

of surface fea-

and

of the vessel is further disorganized

of lipids and other plasmatic augments

evidence

This in turn accelerates

due to the influx

oxygen

demand

and

hypoxia.

These ideas concerning the importance

of a comprehensive

mechanism

cular changes associated

Key words:

the effects of proteins

of the diffusion resistance

transport,

as well as

for the occurrence

of atherosclerosis

and other vas-

with aging.

Atherosclerosis Rabbit

on oxygen

of blood plasma, provide strong indications

- Cholesterol

aorta - Scanning

- Di$zLsion

electron

- Hypoxia

- Plasma

proteins

-

microscopy

INTRODUCTIO&

That deprivation a new theory.

However,

of oxygen is a factor in the initiation it is still unknown

tissue interface.

This study proposes

is very sensitive

to the concentrations

variations

can bring about the hypoxic

that

of atherosclerosis

what causes this hypoxia diffusion

of oxygen

of the plasma proteins; conditions

is not

at the blood-

in the blood plasma and that such protein

for the initiation Atherosclerosis,

of atherosclerotic 1972, 15: 327-343

328

G. M. CHISOLM,J. L. GAINER,G. E. STONER, J. V. GAINER, JR.

change. It will also be pointed is very important the hypoxia

theory,

ture and changes hypothesis,

out that the resistance

in determining parallels leading

the amount

to oxygen diffusion by the plasma

of nutrients

can be drawn between

to edema

a brief review of evidence

to tissue.

Using

senile changes in vascular

struc-

and atherosclerosis.

supplied

As an introduction

for and consequences

of hypoxia

to this

is presented.

BACKGROUND

An extensive review and resulting hypothesis of the hypoxia phenomenon were presented by HUEPERI in the mid 1940’s. He suggests that the common action of agents

causing

vascular

injury

leading

to edema or atherosclerosis

is an interfer-

ence, either short and severe or prolonged and moderate, with the oxidative metabolism of the tissue comprising the blood vessel wall. This injury increases the wall permeability

and allows imbibition

oxygenation and nutrition Some of his evidence riments

in which animals

of plasmatic

matter

which further

of its cellular elements. for this theory stems from findings in low oxygen

atmospheres

of many

developed

impairs

the

earlier expe-

degeneration

of the

vasculature2-4. Many recent studies of experimental atherosclerosis have been performed on rabbits because of the tendency of these animals to develop spontaneous vascular

disease

and

because

this

tendency

is greatly

feeding. Furthermore, the characteristics of the resulting similar to some types of human atherosclerosis. Studies

of the

effects

of prolonged

hypoxia

et a1.5, KIPSHIDGE~ and ASTRUP et al.7 indicate

of edema,

medial

and degenerative

by cholesterol

vessel lesions

on cholesterol-fed

KJELDSEN

thickening

augmented

vascular

rabbits

a pronounced lesions

are quite by

worsening

in lowered

oxygen

atmospheres. These studies indicate the occurrence of an increased permeability of the endothelium and intima to cholesterol, triglycerides and proteins537, as well as impairment of the capacity for oxidation and therefore excretion of cholesterols. Repeated severe exposures to anoxia also appear to cause formation of grossly visible necrotic lesions and vascular injury even in rabbits not being fed a high cholesterol

diets~sJ0.

Increases

in the weight

of the heart

and aorta

as well as the

inner surface area of the aorta have been reported. Disintegration, fragmentation and straightening of the elastic membranes usually associated with increased permeability were very noticeable. CONSTANTINIDESII reports that even though extreme mechanical distension of arterial walls failed to open endothelial junctions to increase permeability, clamping of the vessel causing anoxia opened these junctions in a short period. Macroscopic and microscopic change of a similar nature in the rabbit aorta were found in experiments on cholesterol-fed rabbits breathing carbon monoxide enriched atmospheres that resulted in carboxyhemoglobin concentrations similar to that of heavy smokers7JsJs. These experiments suggest that hypoxia caused by the carbon monoxide increases destruction of the vascular lining, permeability of the endothelium, local edema andsubsequent atheroma. Conversely, exposing cholesterol-fed Atherosclerosis,1972, 15: 327-343

PLASMA PROTEINS,

rabbits

OXYGEN TRANSPOD ATHEROSCLEROSISRT

to a hyperoxic

visible

aortic

des7J4.

lesions

atmosphere and aortic

Furthermore,

radiation

or both

Oxygen reasons. intima

For and

through

ALTSCHUL

decrease

supply

to

example,

portion

one-third

of the inner

arterial

This suggests

and therefore

might

and intimal

A deficiency

lactic

acid

causes

views

affected

changes

xia.

would

then

and inhibit

blood

many the

flowing

of the media

Csing

an average

that this avascular penetration

distance

on the verge

to alterations

Thus,

capacity

of hypoxia

in oxygen

tension

which

demands

patient

where

local

because

excessive

as micellar

dispersion

protein

of globular

fat

to

of the phos-

the lipid

micelles

by the cell. This results in lipid-

hypoxia

16sI9. This

of the narrowing

amounts

lipids

fat. That

further

capacity

are not satisfied,

be mobilized

and accelerates

that local

and local hypo-

can inhibit

the emulsifying

of most

to extracellular

substances

for

than

He proposes

of metabolites

hypoxia

reduces

fat that cannot

or aging

lipidic

as a vicious

accumulation

to be lower

permeability

are necessary

oxygen

intima

transport

wall

excessive

of metabolites.

membrane

cholesterol

vascular

cycle

is intensified

of the vasa vasorum,

of cholesterol

impede

the emulsi-

of the phospholipidsI6.

Although hypoxia,

of the arterial

phosphorylation,

if the increased

to globular

at the

to prevent

of the cells to disperse

as well as in the diabetic

ROBERTSON what

patients

suggests

actually

and how

causes

In fact,

it is not clear why

a particular

since it lies adjacent

“hemodynamic

the local

the endothelium

is still conjecture.

many

to blood

might

investigators

which

react

reflect

part of a normal rich in oxygen.

changes”

changes

as being

bring

to imbibe

Thus we began

hypoxia

plasmatic

the comment

vessel should

responsible

about

in

matter

of RREDT~~ that

be deprived to investigate

of oxygen, the etio-

of such hypoxia. That

blood

suspensions

cells)

is a generally with

is constantly

of hypoxia

reserves

synthesis,

in the hypertensive

logy

on the

calculated

humans. for

is such that

the outer two-thirds

sensitive

can result in reduced

cells or atherocytes

normal

media

by the availability

Furthermore,

are changed

for

in humans

to the limiting

mechanisms

with oxidative

phospholipid fat.

result

cause increased

the ability

is, by interfering

the

the energy

hemodynamic

pholipids.

is critical

vasorunl1,I3,I4,16,17,

close

to be very

of regulatory

and easily

micellar

the inner

be expected

ROBERTsoN1g

This

that

is very

ultraviolet

and in some

arteries

AND LAURSENI~

triglyceri-

hyperoxia,

in rabbits

Only

deposition,

and

thicknessIOJ4~19.

cycle.

tissues,

wall

in lipid

are dependent

of nutrients.

KIRK

that

of the aorta

by the vasa

tissue,

decrease

of larger

media

329

phospholipids

report

levels

tissue

of the

are supplied

rate for aortic

for oxygen.

fying

cholesterol

luminal

the vessel for their supply

respiration

laden

AND HERMAN~~

serum the

a marked

of cholesterol,

the vascularization

the inner

and the adventitia

and

showed

content

AN

tending

accepted

hemoglobin

must

through

a layer

through

the plasma

such as blood

toward

concept diffuse

of plasma

in the larger

the center even

surrounded in pulsatile

not only

on its way

were decreased,

through

arteries

flow.

with

tissue.

hypoxia

particles

sheath

Therefore,

the erythrocyte

to the vascular the resulting

flow

by a peripheral

oxygen membrane

If the oxygen

could upset

~4therosclerosis,

(red

of plasma combined but also transport

the seemingly

1972, 15: 327-343

G. M. CHISOLM,

330 delicate

metabolic

balance

J. L. GAINER,

of the vasculature

G. E. STONER,

and result

J. V. GAINER,

in edema.

Ii.

Therefore,

an

understanding of oxygen transport through blood plasma is necessary in understanding the overall transport process. It has long been noted in the chemical processing of polymers that the diffusion of any substance, concentration through

human

centrations

such as oxygen,

of the polymer plasma,

through a polymer solution is dependent on the present 21. This would suggest that diffusion of oxygen

also a polymer

of the plasma

proteins.

solution,

might be dependent

To determine

this, the following

upon the conexperiment

was

undertaken. A solution

with the same chemical

was developed human stances organic

which contained

and physical

average

values

properties

of the 60-70

as human

known

plasma

substances

of

plasma including proteins, lipoproteins, nonprotein nitrogen-containing sub(e.g., urea, bilirubin, creatine and uric acid), amino acids, carbohydrates, acids,

lipids

and electrolytes.

varied and diffusivities for individual

variations

cholesterol. The results

Then

were measured in albumin,

were strikingss,ss.

the concentration

in a diffusion

gamma-globulin For example,

was varied from 0 to 6.0 g/100 ml, the diffusivity

of one protein

cell apparatus. and

was

This was repeated

fibrinogen,

as the concentration of oxygen decreased

as well as of albumin

by approxima-

tely 60 %, and this decrease occurred over what is usually considered to be the normal physiological range for humans (2.8-4.5 g/100 ml). These results are shown in Fig. 1 for both 25 and 37 “C. Similar results were obtained for other experiments in which the concentrations of gamma-globulin, fibronogen and cholesterol were varied (see Fig. 2). Again

the decrease

o.75Lu

. . g albumin/lOOml

in diffusivity

5.0 6.0 fabricated plasma

of oxygen

with increasing

o.75-___ 0.60 1.20 0.00 0.40 g gamma-globulin/lOOml plasma

Fig.

1. Variation

of oxygen

diffusivity

in plasma

with albumin

Fig.

2. Variation

of oxygen

diffusivity

in plasma

with gamma-globulin

Atherosclerosis.

1972, 15: 327-343

concentration

concentration. concentration

2!z 1.60 fabricated

TRANSPORT

PLASMAPROTEINS,OXYGEN

occurred

over the normal

by showing

that

physiological

the diffusivity

the same as in the fabricated We therefore be responsible more prevalent concentration

AND

began

range.

of oxygen that

age, and found human

increases

were later corroborated plasma

samples

in the plasma

at the vascular

in the aged, we first reviewed with

These results

of real human

behaved

sample.

to postulate

for the onset of hypoxia

decrease as the normal

331

ATHEROSCLEROSIS

that

the variation

the oxygen

proteins

might

wall. Since atherosclerosis of protein

diffusivity

is

and cholesterol

in plasma

gets older. These results have previously

should

been reported

in Nature24.

EXPERIMENTAL

In light of these results a study was performed These were divided rarily

raised

using sixty Dutch-belted

into 6 equal groups. One group had serum albumin

l-2 g/100 ml by intramuscular

injections

of isotonic

rabbits.

levels tempo-

concentrated

al-

bumin solution every lo-14 days for 6 months. A second group had serum gammaglobulin levels temporarily raised to 130-15Ooj, of the normal value by intramuscular injections of isotonic concentrated gamma-globulin in solution every lo-14 days for 6 months. treated rabbit

A third group was used as a control

as those above except that a 1 y0 cholesterol chow. “Spot-check”

performed fractions

during

the experiments

were taken

of albumin

electrophoretic

diet was substituted

determinations

and measurements

for all rabbits

group. The other 3 groups were of plasma

of total

for normal proteins

protein

prior to sacrifice. These showed a gradual

over a two-week period following injection

were

and protein

and a more constantly

decline elevated

value of gamma-globulin. After six months the animals were sacrificed and aortic samples examined using scanning electron microscopy. This method of analysis was chosen in order to follow fine structural changes in the endothelial lining. The method of sacrificing included intravenous injections of sodium pentobarbital and perfusion of the left heart with saline followed by 4% formaldehyde in saline. Aortic samples were immediately transferred ximately

to diluted one square

formalin centimeter

in saline and stored for microscopy. area were taken

Samples of appro-

from the lower thoracic

aorta just

above the abdominal bifurcation. These were later mounted, dried and coated with a 50-100 A layer of gold and viewed in a scanning electron microscope. Representative pictures

were taken

of each sample.

RESULTS

Figs. 3 and 4 show a normal (control) aortic sample at 500 X, 1000 x , and 2000 x . The normal samples confirm the findings of SUNAGA et al.25 who also found that scanning electron microscopy revealed the inner layer of the aorta to be made up of longitudinally aligned “endothelial folds” connected by small fibers or “bridges”. Atherosclerosis,

1972,

15: 327-343

332

G. E. STONER, J. V. GAINER,

G. M. CHISOLM, J. L. GAINER,

JR.

Fig. 3. Normal rabbit aorta (no injections of proteins, regular rabbit chow diet), x 500.

Fig.

4 shows such “bridges”

are visible

in the “valleys”

between

presence of a few red cells on the sample provides a readily available The folds in the vasoconstrictive about

10 microns

across in many instances elongated That

role in vascular

the endothelium

all the more important moving

plasma

apart.

has confirmed

offers the equivalent

resistance

in folds indicates

in diffusion

studies

diffusing

the plasma

of 10 microns

endothelial

rabbit

the plasma layer is

a plasma

similar

layer of only

to that

3

in the systemic

for most of the intravascular

diffusion

or more. which had been injected

chow diet for 6 months.

Note the breaks

in this group and virtually

group. One might view this as the endothelial 1972, 15: 327-343

barrier.

as a red cell membrane.

folds. These “lakes” are smooth areas where the structure

ed. These areas were very prevalent

Atherosclerosis,

that

to oxygen

situation

zone accounts

if it has a thickness and ate a normal

are

that the bridges play

to the blood-tissue

in vitro that

diffusive resistance

Fig. 5 shows two views of an aorta of a rabbit albumin

the “bridges”

since a layer 10 microns deep of slower

by oxygen

He also showed for an oxygen-transport that

folds but also go

state,

permeability. is arranged

must be crossed

microns

circulation

neighboring

SUNAGA et al.25 suggest

in diffusion resistance

SINHA~~

The

10 microns apart and the “valleys”

often connect

to the next fold. In the dilated

and the folds farther

an important

state are about

deep. The “bridges”

the folds.

measure of size.

with in the

has disintegrat-

absent in the control

bridges being destroyed,

leaving the

PLASMA

PROTEINS,

OXYGEN

TRANSPORT

Fig. 4. A: normal rabbit aorta (no injections denoted by arrow in A, x 2000.

AND

ATHEROSCLER

of proteins,

regular

333

OSIS

czliet),

x

1000: R: detail of area

Atherosclerosis,

1972, 15: 327-343

334

G. M. CHISOLM, J. L. GAINER,

G. E. STONER, J. V. GAINER,

Fig. 5. A: Aorta of rabbit having elevated albumin concentration x 100; B: detail of area denoted by arrow in A, x 500.

Atherosclerosis,

1972, 15: 327-343

JR.

(normal r,abbit chow diet)

PLASMA

PROTEINS,

OXYGEN

TRANSPORT

Fig. 6. .4: Aorta of rabbit having elevated area denoted by arrow in .4, x 500.

AND

335

ATHEROSCLEROSIS

gamma-globulin

level (n, x-ma1 diet),

Atherosclerosis,

x

100; R: de1:ail of

1972,

15: 327-343

336

G. M. CHISOLM, J. L. GAINER,

G. E. STONER, J. V. GAINER,

JR.

Fig. 7. A: Aorta of rabbit having elevated albumin levels, and after 6 months on a 1y0 cholesterol diet, x 50; B: detail of area denoted by arrow in A, x 500.

Atherosclerosis,

1972, 15: 327-343

PLASMA PROTEINS,

endothelial

OXYGEN

folds with nothing

corresponding the hypoxia

to hold them together.

to the crosslinking

more permeable

of synthetic

as the number

produced

337

TRANSPORT AND ATHEROSCLEROSIS

of crosslinks

by the elevated

This might

polymer decreases.

protein

be thought

which become

The analogy

would be that

level has made the endothelial

more permeable. Fig. 6 shows two views of an aorta of a rabbit

of as

membranes,

which had injections

lining

of gamma-

globulin and was fed a normal diet for 6 months. Again note the smooth areas, typical of all rabbits in this group, adjacent to the bifurcation. Since for flowing blood there may be a stagnation greater

molecular

zone near a bifurcation27,

diffusion

would be of even

importance there as the mechanism for oxygen transport. Fig. 7 shows the aorta of a rabbit having albumin injections

lesterol

diet for 6 months.

by the elevated into the intima

We have been postulating

and a 1 yO cho-

that the smooth

areas caused

protein levels are where the larger molecular species can be imbibed and the media. In that connection, note the close up of one of the

lesions. The lesion itself appears to be surrounded by a small zone of smooth material, then farther out, the folds reappear. This would happen if lipids and edematous material menon,

were imbibed except

cholesterol

through

the smooth

it is for a rabbit

having

areas. Fig. 8 shows a very similar phenogamma-globulin

injections

and on a 1 y.

diet for 6 months.

Fig. 9 shows the aorta

of a rabbit

having

normal

plasma

protein

levels, but

having been fed a 1 yO cholesterol diet for 6 months. Some lesions were present in such rabbits, but they were of far less severity than those in the rabbits having elevated protein

levels and fed the cholesterol

diet. Another

interesting

feature

of this group

is the fact that there were few “lakes” present as with the specimens having elevated protein levels. In fact, a close-up shows that most of the “endothelial bridges” are still intact. This might be interpreted as demonstrating that, in the case of oxygen diffusivity nearer normal, the permeability of the endothelial lining is affected only at the place where molecular diffusion is most important - at the thicker stagnant zone of plasma where a thinner

near

the bifurcation.

Hence

plasma layer exists. However,

(as in the case of elevated

proteins),

lesions

are not formed

if the diffusivity

then the thickness

at the places

is lowered significantly

of the plasma

layer next to

the wall provides sufficient resistance to affect the overall transport of oxygen, and lesions may form in areas other than immediately adjacent to the bifurcation. Preliminary results of studies on entire aortas made with the assistance of the Department of Pathology, University of Virginia Hospital, show the same sequence of severity in atheroma formation. That is, the rabbits on both raised protein levels and cholesterol diet had a significantly greater percentage of aortic area covered by lesions than those on cholesterol diet alone. The resulting disappearance of folds caused by the breaking of the endothelial bridge may be the correlating link between aging and the incidence of atherosclerosis. As previously mentioned, evidence has been presented that gamma-globulin and cholesterol increase with age in “normal” human subject+. The resulting decline in oxygen diffusivity in plasma might create a prolonged hypoxic condition and a .4 themsclerosis,

1972,

15: 327-343

338

G. M. CHISOLM, J. L. GAINER, G. E. STONER, J. V. GAINER,

JR.

Fig. 8. A: Aorta of rabbit having elevated gamma-globulin level, and after 6 months on a 1% cholesterol diet, x 100; B: detail of area denoted by arrow in A, x 500.

Athevosclwosis, 1972, 15: 327-343

PLASMA PROTEINS,

OXYGEN TRANSPORT AND ATHEROSCLEROSIS

339

Fig. 9. 4: Rabbit aorta. No injections of proteins, but after 6 manlths on a 1% cholesterol diet, x 100; R: detail of area denoted by arrow in A, x 1000.

Atkevosclerosis, 1972. 15: 327-343

G. M. CHISOLM, J. L. GAINER, G. E. STONER, J. V. GAINER, JR.

340

Fig. 10. Cross-section

of normal rabbit aorta showing corrugated

Fig. 11. Cross-section diet, x 500.

of the aorta of a sclerotic rabbit on high-pr otein injections

Atherosclerosis,

1972, 15: 327-343

layers,

X 500.

and cholesl zero1

PLASMA

PROTEINS,

smoothing increased in Figs.

AND

to plasmatic

341

ATHEROSCLEROSIS

10 and 11. Fig.

throughout,

matter,

for the loss of elasticity

the layers

or a tendency

accompanying

the intima

is absent

is demonstrated

rabbit

of the layered aorta behind a vascular level and high cholesterol

and the layers appear straight

and permanently

Fig. 12 shows an aortic sample of a rabbit on high gamma-globulin and separating

Note

appearance

dis-

loss of elasticity.

diet. Here the smooth to embolism,

aorta.

have a corrugated

exposed to raised serum gamma-globulin

diet. The corrugation indicating

of a normal

and media

while Fig. 11 shows a cross-section

in the vessel and

toward edema.

atherosclerosis

10 shows the cross-section

comprising

lesion in a rabbit tended,

TRANSPORT

of the vascular folds. This should cause a loss of elasticity permeability

Evidence that

OXYGEN

area appears

to be flaking

from the deeper vascular layers. This phenomenon in which the fragments

dissociate

emboli in the first small vessel they encounter. the facts that (1) embolism

and a normal

and on the verge of fragmenting may be a precursor

from the vessel wall and lodge as This idea is indirectly

can occur in patients

with no history

supported

of rheumatic

by

fever

as well as in those with an absence of sclerotic lesions or evident vascular injury upon autopsy,

and (2) embolism

with age might diffusivity

occurs more often in the aged as. Thus, increased proteins

cause vascular

fragmentation

and embolism

which, in turn, affects luminal vascular

In the interpretation

of vascular

of protein into a living organism,

Fig. 12. Aortic lumen of rabbit ed endothelium, x 500.

having

elexzatcd protein

plasma

structure.

disorganization

the possibility

by affecting

following

of immune

levels showing

the introduction

response to the protein

fragmentation

d therosclerosis,

of the smooth-

1972,

15: 327-343

G. M. CHISOLM, J. L. GAINER, G. E. STONER, J. V. GAINER, JR.

342 followed by Arthus

reaction

should

be considered.

In recent

years the formation

of

lesions caused by Arthus reaction has been investigated and shown to depend on four factors: Antigen, antibody, complement and polymorphonuclear leukocytes29JO. The reaction

of all four of these components

can cause a local vasculitis.

The vascular

basement membrane and endothelium are damaged, causing local hemorrhage thrombosis. Strong reactions may eventually lead to necrosis30. In the present

study

increases

it is not likely that Arthus

logically

induced

vascular

changes in the high protein

The albumin-injected

in vascular rabbits

reaction

permeability

and related

are responsible

and

immuno-

for the noted

subjects. received

rabbit

serum albumin

(RSA). Therefore,

no antigenic response would be expected from such an introduction of isologous serum proteins. The presence, however, of antigenic response in the rabbits injected with gamma-globulin is possible since the injections were of human gamma-globulin (HGG). That the noted smoothing of endothelial folds is attributable to this response is unlikely because the microscopic vascular changes of the present experiments were the same for rabbits injected with either the isologous RSA or the heterologous HGG. Moreover, the typical symptoms of anaphylaxis in the rabbit - irregular breathing followed by rapid breathing, nystagmus and collapse due to right heart failure30 -

were not encountered

in any of the subjects.

Also, experiments

cited by

COCHRANE

revealed

et aZ.29,in which HGG-anti-HGG complexes were injected into rabbits, no detectable evidence of immune complex deposition in vessels upon histo-

logic and fluorescent antibody examination of tissues removed one to six hours after infusion29. Since it is suspected that the antigen-antibody reaction is humoral and suggest that the tissue damage is a secondary incident 31, COCHRANE’S experiments secondary

uptake

particular

affinity between

of the complexes

by endothelial

HGG-anti-HGG

cells is absent

complexes

and vascular

and there

is no

tissue2Q.

REFERENCES

6 7

s

9

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