Modification of the Effects of Hypertension on Lysosomes and
Connective Tissue in the Rat Aorta
By Harvey Wolinsky, Sidney Goldfischer, Bernice Schiller, and Lisa E. Kasak
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
ABSTRACT
The relationship of lysosomes to the vascular effects of hypertension and the possible
modification of these effects by anti-inflammatory agents (methylprednisolone and
aspirin), vitamin E, and estrogen were studied. Each of these agents was given to a group
of hypertensive rats; untreated hypertensive rats and normotensive rats served as controls. Vessel wall morphology and dimensions of aortas from hypertensive rats were
unaffected by treatment. Usual connective tissue accumulations seen in hypertensive
vessels were suppressed to normotensive levels in the methylprednisolone- and estrogentreated hypertensive groups. Two lysosomal enzymes, acid phosphatase and Nacetyl-/3-d-glucosaminidase (NAGA), increased over normal levels in hypertensive
vessels from 1.03 ± 0.08 (SE) to 2.04 ± 0.19 /imoles/aortic pair hour"1 and from
1.10 ± 0.24 to 4.57 ± 0.38 /^moles/aortic pair hour"1 for the respective enzymes. Normal
enzyme levels in estrogen-treated hypertensive rats (1.14 ± 0.15 /^moles/aortic pair
hour"1 for acid phosphatase and 2.04 ± 0.44 /imoles/aortic pair hour"1 for NAGA) and intermediate levels in methylprednisolone-treated hypertensive rats (1.35 ± 0.10 /tmoles/
aortic pair hour"1 for acid phosphatase and 2.48 ± 0.54 /^moles/aortic pair hour"1 for
NAGA) were found. Other treated groups showed the usual elevations associated with
hypertension. These group differences were also seen after cytochemical staining for
lysosomal acid phosphatase and NAGA. The parallel changes in aortic connective tissue
and lysosomal enzymes in hypertension and their modification by two drugs suggest that
these events are related.
elastin
iV-acetylglucosaminidase
aspirin
vitamin E
KEY WORDS
• Vascular smooth muscle cell proliferation and
connective tissue accumulation are dominant
features of the vessel wall response to a variety of
injurious stimuli (1). These same features are also
prominent during growth and development of the
atherosclerotic plaque (2), which suggests that
tissue injury may be an important component of lesion progression. Lysosomes are closely involved in
the cellular response to tissue injury at many sites
(3), and their activity in vascular disease might
therefore be a useful indicator of the occurrence
and the degree of damage and repair. Modification
of tissue responses to injury, specifically in vascular
tissue, might provide insight into mechanisms by
which injury gives rise to overt disease and offer a
rational basis for intervention (1).
From the Departments of Medicine and Pathology, The
Albert Einstein College of Medicine, Bronx, New York 10461.
This study was supported in part by U. S. Public Health Service Crants HL 12766, NS 06856, and HL 13979 from the National Institutes of Health. Dr. Wolinsky is the recipient of a
Research Career Development Award from the National Heart
and Lung Institute.
Received October 5, 1973. Accepted for publication December 14, 1973.
Circulation Research, Vol. XXXIV, February 1974
collagen
acid phosphatase
estrogen
methylprednisolone
vascular smooth muscle
Apart from its proven accelerating effect on the
development of atherosclerosis (4, 5), hypertension
is a useful model for the study of vascular injury; it
causes cellular hypertrophy (6), proliferation (7),
and degeneration (8, 9) and accumulation of intramural connective tissue (10). Wolinsky (11) has
shown that administration of drugs such as estrogen
significantly inhibits this response to hypertension.
Peters et al. (12) have recently isolated a lysosomal
fraction from rabbit aortic wall, and we (13) have
recently identified the lysosomes in vascular
smooth muscle by ultrastructural cytochemistry.
The present study was intended to explore further
the relationship of lysosomes to the vascular effects
of experimental hypertension and to study possible
modifications of these effects by anti-inflammatory
agents (methylprednisolone and aspirin), vitamin E,
and estrogen.
Methods
Male Carworth (CF-N) rats weighing 170-180 g at
the outset of the experiment were used. Hypertension
ensued 3 weeks after placement of a left renal artery
clip under light ether anesthesia. Systolic blood pres-
233
234
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sures were measured using a tail cuff and a transducer by
an amplification technique (model 79 Grass amplifier);
hypertension was defined as a systolic blood pressure
greater than 150 mm Hg. Rats were given Purina rat
chow and water ad libitum. Six groups of 30 rats each
were used: group N = untreated normotensive rats,
group H = hypertensive rats untreated except for
weekly intramuscular injections (0.05 ml) of the vehicle
(29 mg of polyethylene glycol 4000, 8.7 mg of sodium
chloride, and 0.19 mg of myristyl-y-picolinium chloride
per 1 ml of vehicle)1 for methylprednisolone acetate (40
mg/ml,1 group M = hypertensive rats given weekly intramuscular injections of 2 mg of methylprednisolone
(0.05 ml), group V = hypertensive rats given intramuscular injections of 200 IU of vitamin E (di-a-tocopheryl
acetate, lot 22304, USV Pharmaceutical Corp.) (0.2 ml)
on alternate days, group A = hypertensive rats given
aspirin (Merck) in their chow at a daily dose increased
from approximately 40-60 mg at the outset to 70-100
mg by 3 weeks and maintained at that level thereafter,
and group E = hypertensive rats given weekly subcutaneous injections of 100 jug of estradiol cypionate1 in cottonseed oil (0.1 ml). All drugs were begun 1 week after
clipping (and therefore 2 weeks before the onset of hypertension) and were continued for the duration of the
experiment, which lasted through an 8-week period of
documented hypertension. Blood for serum salicylate
levels was drawn from random group A rats during the
second month of hypertension. Adequacy of salicylate
intake was confirmed by serum salicylate levels which
averaged 16.6 ± 0.6 (SE) mg/100 ml.
After the rats were killed by exsanguination under
ether anesthesia, thoracic aortic segments were excised
for study. The thoracic segment was defined as that portion anatomically delimited by the left subclavian arteries and the celiac artery. Cytochemical studies were
also carried out on the aortic arch of some rats.
MORPHOLOGY
Thoracic aortic segments from four rats per group
were prepared for distention to the blood pressure of
the donor rat as described elsewhere; the distention
mixture contained barium sulfate, gelatin, and carbon
(10). After fixation, diameters were measured on x-rays,
and after paraffin embedding micrometric measurements were made as previously described (10). Circumferential wall tension was calculated according to the
Law of Laplace (wall tension = blood pressure X
radius); wall stress was determined by dividing the tension by wall thickness. All measurements were corrected
for tissue shrinkage during preparation as determined
previously (10).
CYTOCHEMISTRY
Aortic segments, removed immediately after death,
were fixed for 3 hours in ice-cold 2.5% glutaraldehyde in
0.1M cacodylate buffer, pH 7.4 (14). Specimens were
iKindly supplied by Upjohn Company, Kalamazoo, Michigan.
WOLINSKY, GOLDFISCHER, SCHILLER, KASAK
labeled with a code that was not made available to the
tissue laboratory until the slides were processed and reported. After fixation tissues were rinsed in buffer containing 7.5% sucrose, and frozen sections lO/i thick were
cut with a Sartorius freezing microtome for light
microscopic study. Freely floating sections were incubated for acid phosphatase activity (20, 30, and 45
minutes at 37°C) in a modified Gomori medium with /3glycerophosphate as substrate (15) and for N-acetyl-/3d-glucosaminidase (NAGA) activity (60, 75, and 90
minutes at 37°C) in a hexazotized pararosanilin medium
(16) with naphthol AS-BI N-acetyl-/3-d-glucosamine
(Sigma) as a substrate. After incubation, acid
phosphatase preparations were rinsed, visualized in
dilute ammonium sulfide and mounted on slides with a
water-soluble medium. NAGA preparations were
dehydrated and mounted in Permount. For electron
microscopy, frozen sections 40/i thick were incubated
for acid phosphatase activity, as above, rinsed in buffer,
and postfixed for 1 hour in 1% osmium tetroxide in 0.1M
phosphate buffer, pH 7.4 (15). Specimens were then
dehydrated in graded alcohols and embedded in Epon.
Thin sections were cut with a Sorvall MT-2
ultramicrotome and examined in a Siemens Elmiskop.
BIOCHEMISTRY
All studies were carried out on intima-media fragments which were stripped from the adventitia of complete thoracic aortic segments.
Connective Tissue and Cellular Proteins.—Six rats
per group were used, and the intima-media fragments of
an entire aorta were analyzed. After delipidation and
dehydration, soluble and residue fractions were prepared according to a modification of Lansing's method
(10). After acid hydrolysis, total elastin and collagen and
noncollagenous alkali-soluble proteins (NCASP) per
aorta were also determined. The latter component is felt
to represent mainly the cellular protein component of
the vessel wall (10).
Enzymes. — Determinations were carried out on five
pairs of aortas per group. Intima-media fragments from
each pair were placed in distilled water and
homogenized for 6 minutes with a Teflon mortar and
pestle; temperature was maintained at 4°C throughout
this procedure. Centrifugation at 4°C at 1,000 g for 10
minutes was followed by separation of the supernatant
fluid for enzyme analyses; the pellet was used for DNA
determinations. Completeness of this separation was
confirmed by "reverse" analyses of some pellets for
enzyme activities and of some supernatant samples for
DNA; only trace amounts of each were found. Acid
phosphatase activity was assayed using /3glycerophosphate (Sigma) as the substrate and following
the method of Barrett (17) after appropriate reduction
of volumes of reagents. Total NAGA activity was assayed
using the appropriate p-nitrophenyl substrate (Sigma)
and the method outlined by Barrett (17); this method
was modified again for the small amounts of tissue. Time
Circulation Research. Vol. XXXIV. February 1974
235
LYSOSOMES, FIBROSIS, AND HYPERTENSION
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curves were evaluated to determine optimal conditions,
and appropriate reagent and tissue blanks were checked
for base-line activity. Enzyme activities are based on
micromoles of nitrophenol released by the NAGA (18)
and micromoles of inorganic phosphorus released by the
phosphatase, measured using the modified method of
Fiske and Subbarow (19). Activity is expressed in terms
of total micromoles of each substrate released per aortic
pair per hour.
DNA.—Total DNA content of five pairs of aortas from
each group was determined on pellets (see above). The
modification by Hubbard et al. (20) of Cerrotti's method
was used. DNA standards were corrected for water content with inorganic phosphorus as the criterion, using
the method of Fiske and Subbarow (19).
For all statistical comparisons of results, Student's ttest was used. Significance was considered to be present
at the 5% level or less.
Results
Body weights, blood pressures, and heart
weight-body weight ratios of rats in each of the
groups are shown in Table 1. Final body weights
were all similar except for groups M and E, which
were significantly lighter than the others (P< 0.001
for both). Final blood pressure levels (average of
the last two readings taken at weeks 5 and 7) did
not differ significantly among any of the hypertensive groups; all were significantly greater than that
of group N (P< 0.001). Similar findings were seen
for the heart weight-body weight ratios (Table 1),
which reflect cardiac enlargement due to hypertension; all hypertensive groups were similar and
differed significantly from group N (P< 0.001).
MORPHOLOGY
The results of morphometry and tension calculations are shown in Table 2. All the hypertensive
groups in Table 2 had similar aortic diameters
which were all significantly greater than those of
the normotensive rats in group N (P<0.0l). The
same relationship held for each of the other
measurements and calculations shown in Table 2:
the values for the hypertensive groups were similar
to each other regardless of treatment and were significantly greater than those for the normotensive
group. For wall thickness and medial area, the significance of this difference between the hypertensive groups and the normotensive group was
P<0.01; for tension per lamellar unit and wall
stress the significance was P< 0.001. Thus, for all
measurements and calculations made from
morphology and blood pressure, no effects from the
various treatments of the hypertensive groups
were detected.
BIOCHEMISTRY
Biochemical analyses of the aortas, however, did
show differences among the hypertensive groups.
Total aortic dry weight (Fig. 1) for each of the hypertensive groups was greater than that of the normotensive group (P< 0.001) except for group M,
which had an intermediate level heavier than that
of group N {t= 2.63, P< 0.05) and lighter than that
of the other hypertensive groups (P< 0.001).
Wolinksy (11) has previously reported that
estrogen treatment of hypertensive rats reduces
aortic dry weight to normotensive levels, so the
values are not given in this paper.
Analysis of individual components (Fig. 2)
showed that the hypertensive groups H, V, and A
all had significant increases in absolute weights of
elastin and collagen in the aorta compared with
normotensive group N (all comparisons P< 0.001).
However, aortas from methylprednisolone-treated
hypertensive rats (group M) had amounts of these
two proteins which were no different from those in
the normotensive rats U=0.82, 0.5>P>0.4 for
elastin, t=1.74, 0 . 2 > P > 0 . 1 for collagen).
Wolinsky (11) has previously reposed that
estrogen-treated hypertensive rats also r' - not
TABLE 1
Characteristics of Croups Studied
Final body wt
(9)
N
H
M
V
A
E
370
377
268
358
353
277
± 11
± 11
± 10
± 17
± 15
±9
Final systolic blood pressure
(mm Hg)
111
192
195
185
193
179
±1
±7
±7
±5
±8
±6
Heart wt/body wt
(%)
0.28
0.36
0.41
0.37
0.38
0.39
±
±
±
±
±
±
0.01
0.01
0.02
0.01
0.02
0.02
All values are means ± SE. N - normotensive rats, H - hypertensive rats, iM — methylprednisolone-treated hypertensive rats,
V = vitamin E-treated hypertensive rats, A — aspirin-treated hypertensive rats, and E - estrogen-treated hypertensive rats.
Circulation Research, Vol. XXXIV, February 1974
236
WOLINSKY, GOLDFISCHER, SCHILLER, KASAK
12.0
5.0 r
J Meant S.E.
10.0
O)
IGHT
_E
8.0
U-l
6.0
AORTIC
Q
4.0
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2.0
N
H
M
V
FIGURE 1
Total aortic dry weights of the groups studied. All of the values
for the hypertensive groups are significantly greater than that
for the normotensive group (group N) except for the value for
group M, which had an intermediate weight.
differ from normotensive rats in the levels of these
proteins in their aortas.
With respect to NCASP, which mainly reflect
cellular proteins, each of the hypertensive groups,
including group M, had a similar amount of
NCASP—an amount far greater than that in the
normotensive group (P< 0.001). Estrogen-treated
hypertensive rats have previously been shown to
have NCASP levels intermediate between those
found in the other hypertensive rats and the normotensive rats (11).
Enzymes.—Cytochemical studies showed that
FIGURE 2
Weights of individual aortic components in each of the groups
studied. Groups H, V, and A had significantly more collagen
and elastin than did group N; group M did not differ from group
N in amount of connective tissue. All hypertensive groups (H,
M, V, and A) had significantly more noncollagenous alkali-soluble proteins (NCASP) than did the normotensive group (N).
only a few lysosomes could be stained in the medial
smooth muscle cells of aortas isolated from normotensive rats and incubated for acid phosphatase
(45 minutes) or NAGA (90 minutes) activities (Fig.
3a). In aortas of untreated hypertensive rats,
numerous lysosomes were readily demonstrable in
the perinuclear cytoplasm after relatively brief incubations of 30 minutes for acid phosphatase activity and 60 minutes for NAGA activity (Fig. 3b).
TABLE 2
Dimensions of and Calculated Stresses on the Aortic Wall
N
Diameter (mm)
Wall thickness (mm)
Tension/lamellar unit
(dynes/cm x 103)
Wall stress (dynes/cm2 x 106)
Medial area (mm2)
M
2.23 ± 0.08
0.883 ± 0.024
2.68 ± 0.04°
1.140 ± 0.048*
2.60 ± 0.03°
1.172 ±0.058°
2.58 ± 0.06°
1.120 ±0.026°
2.75 ± 0.07°
1.163 ±0.051°
2.03 ±0.11
1.85 ± 0.09
0.646 ± 0.037
4.20 ± 0.16f
2.89 ± 0.07f
4.44 ± 0.29t
2.87 ± 0.08f
3.84 ± 0.18t
2.71 ± O.lOt
0.999 ± 0.053*
1.023 ±0.056"
0.945 ± 0.010°
4.05 ± 0.13t
2.71 ±0.12t
1.047 ± 0.058°
All values are means ± SE. N — normotensive rats, H — hypertensive rats, M - methylprednisolone-treated hypertensive rats,
V = vitamin E-treated hypertensive rats, and A — aspirin-treated hypertensive rats.
°P<0.01 (paired analysis) compared with group N.
tP< 0.001 (paired analysis) compared with group N.
Circulation Reseanh,
VoL XXXIV, February 1974
237
LYSOSOMES, FIBROSIS, AND HYPERTENSION
FKHJRE3
Downloaded from http://circres.ahajournals.org/ by guest on June 17, 2017
AH Jfg/if micrographs (a, b, d, and e) are
of 10fi
frozen
sections
of
glutaraldehyde-fixed rat thoracic aortas
incubated
for
N-acetyl-f3-dgjucosaminidase activity. The electron
micrograph (c) is of a 40fi frozen section incubated for acid phosphatase activity and processed for ultrastructural
examination, a and b: Only a few reactive lysosomes (arrows) are barely
detectable, even after 90 minutes of incubation, in the normal aorta (a). In the
hypertensive
vessel (b) reactive
lysosomes (arrows) are abundant in the
perinuclear cytoplasm of medial smooth
muscle cells after 60 minutes of incubation, c: Electron micrograph of hypertensive rat thoracic aorta. Reaction
product is deposited on lysosomes (L).
A lysosome of the autophagic vacua e
type (AV) is also
identified.
Mitochondria (M) are unreactive.
Clusters of lysosomes are common in
hypertensive vessels but are rarely seen
in the normotensioe vessel, d and e: Oil
immersion photomicrographs show
clusters of darkly stained lysosomes in
the aspirin-treated hypertensive rats (d)
but not in the estrogen-treated hypertensive rats (e).
Circulation Ratarrh, Vol. XXXIV. February 1974
238
WOLINSKY, GOLDFISCHER, SCHILLER, KASAK
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Identification of the reactive sites as lysosomes was
confirmed by examining acid phosphatase preparations in the electron microscope. The product of
the lead phosphate reaction Was deposited on
lysosomes of the dense body and autophagic
vacuole types (Fig. 3c).
Aortas from hypertensive rats treated with
aspirin reacted in the same fashion as those from
the untreated hypertensive rats (Fig. 3d); the reaction of vitamin E-treated hypertensive vessels was
also similar. Aortas from hypertensive rats treated
with estrogen closely resembled those from the
normotensive controls in their paucity of acid hydrolase-reactive lysosomes (Fig. 3e). Aortas from
methylprednisolone-treated hypertensive rats had
more reactive lysosomes than did those from normotensive rats but far fewer than those from untreated hypertensive rats. These group distinctions
were more pronounced in NAGA than in acid
phosphatase preparations.
Results of analyses of aortas for two lysosomal
enzymes are shown in Figure 4. Compared to normotensive rats (group N), untreated hypertensive
rats (group H) had significantly greater activities of
both acid phosphatase and NAGA (t = 4.85,
P< 0.001 and t=7.68, P< 0.001, respectively).
Aspirin-treated hypertensive rats (group A) had increased activities similar to those of untreated hy-
I 7-°
IX
u
6.0
Discussion
B £ ) ACID PHOSPHATASE
0 2 3 NAGA
I
O
Q
5.0
i
4.0
MtontS.E.
o
>•
X
UJ
3.0
5
J—
t/>
CO
pertensive rats. Vitamin E-treated hypertensive
rats (group V) showed marked increases in both
enzymes, significantly greater even than those
found in group H (t=2.58, P<0.05 and f=3.98,
P < 0 . 0 1 , respectively). Methylprednisolonetreated (group M) and estrogen-treated (group E)
hypertensive rats both showed suppressed levels
compared with those found in untreated hypertensive rats (group H). In the case of group M, acid
phophatase was at an intermediate level, very significantly less than that in group H (t=3.19,
P < 0.02) and significantly more than that in group
N (t = 2.82, P < 0.05). NAGA levels in group M were
also very significantly less than those in group H
U=3.15, P<0.02) but were not quite significantly
above those in group N (*= 2.16, 0.1 > P> 0.05). In
group E, activities of both enzymes were suppressed to levels no different than those for normotensive rats (t=0.67, 0.6>P>0.5 for acid
phosphatase, t = 1.65, 0.2 > P> 0.1 for NAGA).
DNA.—Determinations of total DNA in aortic
pairs showed no significant differences between
any of the groups, hypertensive or normotensive.
The actual values expressed as y per aortic pair
(mean ± SE) were: group N 35.4 ± 1.3, group H
36.0 ± 1.7, group M 34.6 ± 2.6, group V
39.5 ± 2.1, group A 37.2 ± 3.4, and group E
34.5 ± 1.4.
k
2.0
o
1.0
FIGURE 4
Acid phosphatase and N-acetyl-fi-d-glucosaminidase (NAGA)
activities in aortic pairs from each of the groups studied.
Groups H, V, and A had significantly more activity of both
enzymes than did group N. Methylprednisolone-treated (group
M) and estrogen-treated (group E) hypertensive rats showed
suppressed activity, group M to intermediate levels and group E
to normotensive levels.
We found parallel changes in connective tissue
accumulation and lysosomal enzyme activity in hypertensive rat aortic walls. These changes were not
related to either cell number (DNA) or cell bulk
(NCASP). Treatment with estrogen and
methylprednisolone (groups E and M) suppressed
both the connective tissue and the lysosomal
responses in hypertensive rats but did not affect
aortic DNA levels. In these rats, NCASP was also
comparable to (group M) or reduced (group E)
relative to the levels in other hypertensive rats.
Treatment of hypertensive rats with aspirin and
vitamin E had little or no effect on cell number,
bulk, lysosomal acid hydrolase activities, or connective tissue accumulations.
Before proceeding with a discussion of these
findings, the possible objection that suppression of
vascular connective tissue seen in the aortas of
groups E and M reflected their slower growth rate
(Table 1) must be considered. Hypertension was
originally chosen as our experimental model,
because we could show that the vascular response
to increased pressure was far greater than any
Circulation Research, Vol. XXXIV, February 1974
LYSOSOMES, FIBROSIS, AND HYPERTENSION
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vascular effects from changes in body weight of the
magnitude found in this study. Severe hypertension
alone might cause retarded body growth (Table 2,
ref. 21) and increased aortic connective tissue. Dissociation of growth and vascular effects has also
been shown in studies of drugs, such as progesterone, which suppress growth but do not affect
the vascular effects of hypertension (11). In a recent study that used controlled feeding, Wolinsky
(22) has shown that the vascular effects of estrogen
administration to normotensive oophoreetomized
rats occur independently of its effects on body
growth. Optimally, possible body growth effects on
aortic composition in groups E and M of the present
study should have been controlled by pair-feeding
of all groups. Nevertheless, we believe that the evidence suggests that the major findings described in
the paper are not growth related.
If one considers the association of increased
lysosomal enzyme activity with connective tissue
accumulation to be other than fortuitous, several
possible mechanisms, which are not necessarily exclusive, might be invoked to relate these events in
the aortic wall. Permeability of the intima to a
variety of plasma constituents (9, 23-25) is
enhanced in the hypertensive vessel. Exposure of
the mural smooth muscle cells to an influx of proteins (24) and lipids (25) that is far in excess of normal should result in increased endocytosis and intracellular digestion within secondary lysosomes.
Circulating proteins and lipids are conveyed into
the cytoplasm by endocytic vacuoles that fuse with
lysosomes, and their contents are subsequently
degraded by the lysosomal acid hydrolases (26). A
direct destructive effect on the lysosome by a specific lipid (27) or protein (7) serum component or
an overwhelming of the cellular disposal system
(28) could result in cell degeneration and necrosis.
Release of cellular and lysosomal contents into the
vessel matrix would then perpetuate the lytic process and elicit further proliferative and reparative
responses.
In addition to the response of lysosomes to endocytosis and their involvement in degenerative
aspects of injury, deDuve (26) has suggested that
they may be activated during repair of damaged
tissue, including connective tissue synthesis. The
accelerated connective tissue and mucopolysaccharide synthesis seen in the hypertensive vessel in
response to increased tension (1) would therefore
necessitate increased metabolic and lysosomal activity. Whether the stimulus to lysosomal activity is
enhanced endocytosis, increased synthesis, or both,
Circulation Reiearch. Vol. XXXIV. February 1974
239
the background of excessive mechanical stress
would further serve to increase cell degeneration
and death.
Of the drugs administered, aspirin and vitamin E
showed little or no effect; estrogen and
methylprednisolone showed distinct suppressive
effects on both lysosomal enzymes and connective
tissue accumulation in hypertensive vessels. The
different findings for aspirin and methylprednisolone suggest that nonspecific anti-inflammatory
activity of these agents cannot be implicated in
these effects on the vessel. Glucocorticoids are
believed to be potent stabilizers of membranes,
particularly those of lysosomes (29). Estrogen has
also recently been shown to inhibit release of
enzymes from lysosomes of polymorphonuclear
leukocytes (30) and to stabilize artificial lipid
spherules (29); in contrast, aspirin has little or no
effect on lysosomal stability (31). Vitamin E in high
doses (as given in this study) is thought to be a
lysosomal membrane labilizer (32). It is of interest
that testosterone, also considered a lysosomal
labilizer (29), has been shown by Wolinsky (33) to
result in increased vascular connective tissue when
it is administered to normotensive male rats. Drug
effects on membrane stability are obtained from in
vitro systems; the extent to which these results are
transferrable to in vivo circumstances is a point of
active controversy. Evidence that destruction of
lysosomal membranes and subsequent leakage of
hydrolases into the cytoplasm leads to tissue necrosis and repair has been obtained in studies of silica
toxicity (34) and gouty arthritis (35).
It is also possible that the effective drugs exert
their primary moderating action not on the
lysosome but rather on vascular permeabilityReduction of endothelial permeability by estrogen
(36) or corticosteroids (37) might result in
decreased needs for phagocytosis and secondary
lysosomal stimulation. Other alternative sites of
drug action undoubtedly are possible.
Somewhat analogous to the findings in this paper
are those by Curreri et al. (38), who showed that
lysosomal enzymes are markedly increased in
atherosclerotic plaque-laden aortic tissue of rabbits fed an atherogenic diet. Administration of cortisone in addition to the diet aggravated the hypercholesterolemia of these rabbits but resulted in
complete protection from disease and maintenance
of activities of lysosomal enzymes at normal levels.
From that experiment and from ours, it appears
that despite the continued presence of hypercholesterolemia or hypertension —two of the
240
WOLINSKY, GOLDFISCHER, SCHILLER, KASAK
most potent risk factors for development of
atherosclerotic vascular disease (5)—it is possible
to modify certain aspects of the vascular response
to those stimuli. Although their precise role remains to be determined, it seems that lysosomes are
an important element of that response.
try and electron microscopy: Preservation of cellular
ultrastructure and enzymatic rctivity by aldehyde fixation. J Cell Biol 17:19-58,1963.
15.
16.
Acknowledgment
The authors thank Dr. Olga O. Blumenfeld and Dr. Marie M.
Daly for their assistance with methodology, Mr. Richard
D'Eletto for his technical help, and Mrs. E. Fay Ricksy for her
efforts on the manuscript.
17.
References
19.
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2.
3.
4.
WOLINSKY, H.: Mesenchymal response of the blood vessel
wall: Potential avenue for understanding and treating
atherosclerosis. Circ Res 32:543-549. 1973.
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SCOTT, R.F., JONES, R., DAOUD,
21.
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Modification of the Effects of Hypertension on Lysosomes and Connective Tissue in the Rat
Aorta
HARVEY WOLINSKY, SIDNEY GOLDFISCHER, BERNICE SCHILLER and LISA E.
KASAK
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Circ Res. 1974;34:233-241
doi: 10.1161/01.RES.34.2.233
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