Modulation of tissue fibrinolysis from hypoxia and hyperoxia

THROMBOSIS RESEARCH 38; 129-136, 1985 0049-3848/85 $3.00 + .OO Printed in the USA. Copyright (c) 1985 Pergamon Press Ltd. All rights reserved.

MODULATION

OF

TISSUE

8. Department

(Received (Received

in

FIBRINOLYSIS

Risberg

and

of Surgery University of

FROM

B.

HYPOXIA

AND

HYPEROXIA

Stenberg

I, Sahlgrenska sjukhuset, Gbteborg, Sweden.

19.1.1984; Accepted in revised form 20.9.1984 by Editor P. Olsson) final form by Executive Editorial Office 14.1.1985)

ABSTRACT The

effects

from

alterations

in

inspired

oxygen

tension

on

the

fibri-

nolytic activity in vessel walls was studied in rats. Tissue plasminogen activator activity was measured semiquantitatively with the histochemical technique. During severe hypoxic conditions (FI02 = 0.05) the fibrinolytic activity in the aorta was significantly reduAt 24 hours with slightly higher ced as compared to control animals.

When breath(FIO = 0.08) the activity was increased. (FIZ ? ? 1.0) the fibrinolytic activity was increased in the aorta after 82hours. Following chronic hypoxia for 6 weeks (FI02 = 0.1) the activity in the caval vein was increased. Changes in inspired oxygen tension thus affected the endothelial cells and chang-

oxygen

tension

ing pure oxygen

ed

their

expression

of

plasminogen

activator

activity.

INTRODUCTION Factors regulating synthesis and release of plasminogen activator (PA) PA is found in many different kinds of cells such as are only partly known. mesothelial cells, epithelial cells and leucocytes, (I, 2). In the vascular system PA originates from the endothelial cells. Many different kinds of stimuli can release PA from the vessel walls. Venous stasis release PA into the blood with progressive reduction in tissue activity (3, 4, 5). The PA activity may

depend on the phase of cellular degeneration or regeneration (6). At local level catecholamines have been implicated in PA-release. Biggs et al. (7) demonstrated that adrenaline increased blood fibrinolysis. Further studies indicated that beta-adrenergic receptors were involved (E), but conflicting

results

have

Alpha-blockade adrenaline (IO).

is

been

reported

ineffective

with in

beta-blocking

preventing

the

agents

In isolated perfused organs catecholamines stimulated response that was inhibited by beta-blocking agents (11). stimulating effect does not seem to be mediated by cyclic vasoactivity from different substances has been implicated

Key

words:

Hypoxia,

hyperoxia,

plasminogen

129

activator,

(9).

stimulating

effects

fibrinolysis,

from a

The fibrinolysis (AMP) (12). The as the release

vessel

wall.

ini-

130

HYPOXIA, HYPEROXIA

tiating

ssin)

mechanism which lacks

& FIBRINOLYSIS

but the strong release vasoactive properties

Vo1.38, No.2

after DDAVP (desaminoargininvasoprecontradicts this hypothesis (13).

DDAVP has no effect in isolated organs (14, 15), a fact hypothesis from Cash of hypothalamic hormonal mediation lysis (16, 17). ing

the

The increased release stimuli such as stasis

that may support the of increased fibrino-

of PA from vascular endothelium (13, 18) is followed by reduced

into blood followtissue activity in

vessel walls (5, 19). vessel walls following possible contributing

The mechanisms behind the reduced tissue activity in prolonged stasis remains speculative. Hypoxia is factor. Clarke & Clifton (1962) ('20) found correlation between decreasing oxygen tension and release of PA. Influence of local milieu factors on fibrinolysis was previously found following aorta-caval the one

transposition in ments interposed

rats (21). Increased in the caval vein.

PA

activity

was

found

in

aortic

seg-

Hypoxia might be one factor operative during the experimental conditions mentioned above and in the present paper we are presenting data from acute and

chronic

effects

of

hypoxia hyperoxia

on in

PA-activity short

in

term

the

vessel

walls

and

furthermore

the

experiments.

MATERIAL

AND

METHODS

Animals (range

Onehundredeleven Wistar rats of both 150-250 g) were used in this study.

the experiments. During rats had free access of Hypoxia We used a specially

mosphere was controlled.

sexes

with

a mean

The rats were not long-term experiment with controlled food and water. designed Three

cage

different

where

the

levels

oxygen of

oxygen

weight

of

200

g

fasted before environment the

content

in

the

concentration

atin

- 0.05, 0.08, 0.10) were used. inspired air (FIO Eig ILt rats were exposed for 30 minutes and IO rats for 2 FI02 = 0.05. hours. For the longer periods of exposure the oxygen concentration had to be increased to permit survival of rats. FI02 = 0.08. Eight rats were exposed for 8 hours and 8 rats for 24 hours. FIO = 0.10. Eight rats were exposed during six weeks. Pre I.iminary studies indicated that using lower FIO than 0.10 was related to high mortality during the first week. At FIO = 6.10 all rats survived. After different exposure times the rats were k!lled. A segment of the infrarenal abdominal aorta was excised and soaked in normal saline to remove blood. The specimens were then immediately frozen in carbon-dioxide snow and stored at -7O’C until analyzed. In the 6 weeks experiment a segment of the abdominal infrarenal caval vein was also excised. Hyperoxia Using the same cage as above rats were exposed to 100% oxygen (FI02 ? ? 1.0). Thirtythree rats were used in this experiment and groups of 7-10 rats were followed for 30 minutes, 2 hours, 8 hours and 24 hours. At end of exposure times the rats were killed and a segment of infrarenal abdominal aorta The specimens were soaked in normal saline and rapidly frozen was excised. at 70°C. Controls To each group of experimental animals 4 normal rats (total number 36) had their corresponding vessels excised. The biopsies from these rats were processed simultaneously with corresponding experimental rats. Histological determination of PA-activity PA-activity in the vessel walls was analyzed using Todd’s histochemical method (22). Seven urn-thick frozen sections of the vessels were covered with the

Vo1.38,

HYPOXIA,

No.2

a plasminogen

enriched

fibrin

HYPEROXIA

film.

periods of time from 15-60 minutes. fibrinolysis were seen as unstained was estimated semiquantitatively in in nolytic zones (21). The activity

131

& FIBRINOLYSIS

The sections were Following fixation

incubated f.or different and staining areas of

zones in the bluish film. The PA-activity 4 degrees based on the sizes of the fibrithe specimens from experimental animals

from control animals. Experimental and were compared to corresponding values simultaneously and the microscopic evaluacontrol specimens were processed tion was made on coded samples. Statistics Statistical evaluation was made using the Wilcoxon rank sum test.

RESULTS from one control biopData are presented in figures 1 and 2. A specimen s) was always processed simultaneously with a specimen from the correspondinq experimental biopsy. The differences in activity between control and experimental animals are presented. Breathing air with 5 per cent oxygen (FI02 ? ? 0.5) for 2 hours significantly reduced the fibrinolytic activity in the aorta as compared to control animals (p < 0.01). Following hypoxia with FI02 = 0.08 as significant increase in activity was found at 24 hours (p < 0.05). (Fig. 1). F102 ? ? 0.10 for 6 weeks did not significantly change the activity in the aorta but a significant increase of PA-activity 0.05). The differences from the control

was

values

found

in

in

this

the

caval

group

were

vein

1.1

$p

-

<

1.2

for

the vein. Breathing 100% oxygen for 8 hours significantly increased PA-activity (p < 0.05). (Fig. 2). At 24 hours the activity was still increased though

nnt

statistically

significant.

F102=0.05

FlO2=0.08

30'

8h

A FIB ACT AORTA

2h FIG.

24h

1

Differences in fibrinolytic activity between control and experimental There was a significant reduction in activity afbiopsies in arbitrary units. ter 2 hours at FIO2 t 0.05 (p < 0.01). Following prolonged exposure to FL0 -lU.08 increased actlvlty was found which was statistically significant at 2 2 -hours (p < 0.05).

HYPOXIA, HYPEROXIA & FIBRINOLYSIS

132

A

FIB ACT

Vo1.38, No.2

4

AORTA x 2 SE F 102 = 1.0

30’

Differences in fibrinolytic biopsies following FI02 = 1.0. creased (p < 0.05).

2h

8h

24h

FIG. 2 activity between control and experimental The activity at 8 hours was significantly in-

DISCUSSION In this study we were primarily interested in the short term effects of anoxia and hyperoxia on the fibrinolytic activity in the vessel walls. Hyperoxia had a stimulating effect on PA-activity after 24 hours. Following hypoxia an elevated activity after 24 hours was preceded by a significant reduction in the activity at 2 hours. The concept that 02-tension can influence release of PA is old (23). Todd (24) speculated on the role of anoxia on fibrinolytic activity and found increased release of PA in ischemic legs. Clarke and Cliffton (20) demonstrated an oxygen tension depending release of PA in obstructed veins. The endothelium is sensitive to changes in the local milieu. Stasis and with blood flow, pressure and composition of the ischemia will interfere blood. The 02-tension will change. Hypobaric hypoxia caused a structural remodelling of the pulmonary endothelial cells with hypertrophy and edema in the subendothelial layer (25). These authors found structural changes in the smooth muscles cells in the pulmonary vessels. The morphological changes might thus participate in the hypoxic vasoconstrictor response in the lungs. Changes in cellular ultrastructure have previously been demonstrated after hypoxia and the signs characterizing the “point of no return” have been fairly well documented (26). Reduction in oxygen delivery to the cells will hamper the oxidative enzymatic processes in mitochondria. Hypoxia will thus be followed by deterioration of most cellular functions. As synthesis and release of PA is one aspect of cellular function it is thus conceivable that following severe hypoxia there is reduced PA activity in the endothelial layer. In isolated perfused lungs hypoxia released plasminogen activators (27). FolJ7:1ing prolonged exposure there seems to be an adaptation to the hypoxic state. In vitro studies of aortic vessel walls have shown that short term anoxia produced gross ultrastructural changes in the endothelial cells (28). In our experiment we had to change the inspired oxygen concentration because at FI02 = 0.05 the rats would not survive beyond the first hours. To permit survival of the rats for 24 hours the FIO, had to be increased to 0.08. Our rats did

HYPOXIA,

Vo1.38, No.2

not

survive

6 weeks exposure

this

oxygen

experiments times

concentration

FI02

might

HYPEROXIA

had

than

to

not

be

be

133

& FIBRINOLYSIS

either

for

increased

to

completely

prolonged 0.10.

time

The

comparable

rats

due

and

for

with

to

the

the

different

altered

The changes in fibrinolytic activity that we previously registered fol owing stasis with progressive reduction in activity (28) could thus part-Fro?. ly be due to changes in OZ-tension. Oxygen toxicity is a wellknown clinical finding with the lung being a vulnerable organ. The toxicity of oxygen is associated with formation of oxygen derived radicals. After oxygen exposure morphological changes have been described in the endothelial cells (30). Following oxygen exposure Takala and Ninnikoski (31) found release of lysosomal enzymes from the lung reflecting cellular damage. Crapo et al (32) examined the effects of FIOZ 1.0 and 0.85 on rat lungs. They found pronounced injuries in the pulmonary capillary endothelium after 60 hours at FIOZ I 1.0. With exposure times less than 40 . Using 85% hours they found no morphological changes in the endothelium oxygen the authors found adaptative changes characterized by hypertrophy of the endothelium. They also found an increased activity of superoxide dismutase as a sign of structural adaptation. One consequence of endothelial damage is an increased capillary permeability in lungs. (33). Following hyperoxia endothelial cells in culture had reduced angiotension converting enzyme (ACE) activity and hence increased release of PGIZ and TXA2 following bradykinin stimulation (34). Lung damage following hyperoxia can be mediated through oxygen radical formation (35). Metabolic disturbances have been found such as reduced serotonin clearance (36) and reduced metabolism of vasoactive hormones (37). Extensive damage was found in endothelial and alveolar epithelial cells with interstitial edema in rabbits exposed to oxygen. Endothelial damage was indicated exposure

by reduced (38). Other

angiotensin aspects of

prostaglandinproduction (39, 40). functional

and

converting enzyme activity after 40 hours the metabolic activity in the lungs such as

metabolization

are

affected

The increased oxygen tension is likely changes in endothelial cells in other

as

well

by

hyperoxia

to cause structural organs than lungs.

and

From our experiments it can not be concluded whether the changes in PAactivity in the aortic wall following hypoxia/hyperoxia were expressions of celltoxic effects or metabolic compensation phenomena. Further studies are needed.

ACKNOWLEDGEMENT. This work was Council (00660).

supported

by

grants

from

The

Swedish

Medical

Research

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