From the Department of Medicine, VA Medical Center, and the University of
Minnesota (Minneapolis).
Correspondence to Geza Simon, MD, Hypertension Clinic, VA Medical Center, Minneapolis, MN 55417.
The second aim of this investigation was to explore the relationship
between the onset of hypertension and the development of structural
vascular changes in Ang II–treated rats. Structural vascular changes
are the hallmark of chronic hypertension, and increased wall-to-lumen
ratio (W/L) of resistance arteries is the predominant
lesion.4 5 6 7 The increase in relative
"thickness" of resistance arteries is responsible for the
"amplifier" property of the arterial circulation in
hypertension, which functionally manifests itself as pressor or
vasoconstrictor hyperresponsiveness. Whether structural vascular
changes are primary or secondary to blood pressure (BP) elevation
remains an unsettled issue. In the majority of studies of hypertension,
there has been a direct relationship between BP and W/L of arteries,
but discrepancies also exist.7 In some
experimental models, there is hypertension without structural vascular
changes, or structural vascular changes are modest in magnitude despite
severe hypertension. Certain strains of rats are more prone to develop
structural vascular changes than other strains.8
Other studies have raised the question of whether a trophic stimulus
alone may be sufficient to induce structural vascular changes and
thereby initiate the hypertensive process.9 Ang
II has been a prime candidate for such a role. These findings suggest
that BP is not the sole determinant of structural vascular changes. In
the present study, therefore, a dissociation was sought between the
extent of Ang II–induced hypertension and the W/L of resistance
arteries as evidence that mechanisms other than the level of BP play a
role in the pathogenesis of structural vascular changes. Finally,
because dietary sodium supplementation potentiates both the
vasoconstrictor and vascular trophic action of Ang
II,10 we also investigated the effect of high
sodium diet on the rate of onset and extent of hypertension and
structural vascular changes in Ang II–treated rats.
Preparation of Rats
Tail Systolic BP Measurements
Intra-Arterial BP Measurements
At the completion of direct BP measurements, arterial blood
samples were obtained from 7 awake rats receiving 100 ng ·
kg-1 · min-1 Ang
II SC and from 15 control rats. Plasma Ang II concentrations (pg/mL)
were measured in Dr Ben Zimmerman's laboratory (Department of
Pharmacology, University of Minnesota) using high-performance
liquid chromatography fractionation and
radioimmunoassay.15
In Situ Tissue Fixation
Morphometric Measurements
For further characterization of structural vascular changes induced by
Ang II treatment, four 2nd-order mesenteric arteries (250 to 350
µm OD) were removed from each of nine rats treated with 100 ng
· kg-1 · min-1
Ang II for 12 weeks and from seven 12-week control rats. The arteries
were cleaned of adhering tissue, dehydrated through graded series of
ethanol, and embedded in epoxy resin. Cross sections of 1 µm
thickness were stained with toluidine blue. Arterial
segments sectioned tangentially (nonuniform thickness of the vessel
wall) were discarded. A video camera (Cell Analysis Systems
Inc) connected to an image analysis processor (NIH Image 1.54,
Public Domain Software) and microcomputer was used to measure lumen
diameter, wall thickness (from endothelium to outer
media), the number of smooth muscle cell layers, and cell thickness.
The latter 3 parameters were measured at 4 points where
arbitrarily drawn horizontal and vertical axes intersected the vessel
wall. Cell thickness was measured at the midpoint of the nucleus along
the short axis of the smooth muscle cell. Measurements obtained from 2
to 4 arteries per rat were averaged. W/L was calculated.
Statistical Analysis
Statistical analysis of small and intermediate-size artery
dimensions was restricted to W/Ls because sampling bias could not be
excluded, considering that measurements were made on only
cross-sectionally cut arteries. Random sampling may result in the
measurement of different size arteries from rat to
rat.7 Also, these arteries were categorized on
the basis of their diameter, which precludes meaningful comparison of
their overall dimensions. W/Ls are relatively unchanged despite
differences in lumen diameter as long as a small range of diameters is
being measured (as in the present study) and are therefore less
affected by sampling bias than measurements of external and lumen
diameter or wall thickness.7 W/Ls in the 7 groups
of variously treated rats were compared with those of control rats by
2-factor ANOVA (factor 1, treatment; factor 2, small- and
intermediate-size arteries) followed by preplanned contrasts
(Superanova). To compare the dimensions of large arteries of rats
treated with 100 ng · kg-1 ·
min-1 Ang II for 12 weeks and of matched control
rats, the 2-factor ANOVA was extended to include the large arteries. In
Ang II–treated rats, the contribution of dose and duration of
treatment to changes in W/L was tested with 2-factor ANOVA (factor 1,
dose; factor 2, 4 or 12 weeks of treatment). For this analysis,
only rats on a normal sodium diet (0.7% NaCl) receiving 50, 100, or
200 ng · kg-1 ·
min-1 Ang II for 4 or 12 weeks were included.
One-way ANOVA was used to compare plasma Ang II concentrations in rats
receiving 100 ng · kg-1 ·
min-1 Ang II SC for 4 weeks with those of
control rats. Null hypotheses were rejected at P<0.05.
The W/Ls of small and intermediate-size mesenteric resistance arteries
of rats in the various treatment groups are summarized in Figures 3
Like the increase in W/L of small arteries of rats treated with 50
ng · kg-1 ·
min-1 Ang II for 12 weeks, the increase in W/L
of small arteries of rats treated with 2% NaCl diet for 12 weeks was
of borderline statistical significance (P<0.07).
Interestingly, 9 of the 17 rats displayed an increase in W/L (10.7%,
mean), whereas the remaining 8 rats had no response at all (W/L=8.5%,
mean). When the 2% NaCl diet of rats was combined with the
administration of 50 ng · kg-1 ·
min-1 Ang II for 12 weeks, a highly significant
increase in W/L was achieved (synergism).
Vessel dimensions of rats treated with 100 ng ·
kg-1 · min-1 Ang
II and of control rats are shown in the
Table
A high degree of correlation was found between directly measured MAP
and the average weekly tail SBP of rats (Figure 6
Previously, we have investigated small and large mesenteric artery
structure in rats treated with various doses of Ang II for 6
weeks.3 We found that the earliest changes were
detectable in the smallest arteries (50 to 100 µm OD), some of
which may be best characterized as arterioles. Because of
methodological problems, morphometric measurements seldom have been
performed on these arteries.4 7 Therefore, their
importance in the development of hypertension may have been overlooked.
Neonatal sympathectomy had a different effect on the
development of structural changes in small and large arteries of rats
treated with 200 ng · kg-1 ·
min-1 Ang II SC for 4 weeks; the increase in W/L
of large arteries was markedly attenuated, along with the attenuation
of hypertension, but that of small arteries was
unaffected.18 These findings suggested that a
direct trophic effect of Ang II played an important role in the
development of structural changes in small arteries, whereas in large
arteries structural changes resulted mainly from elevation of BP and
sympathetic stimulation.
The findings of the present study provide additional evidence that
the earliest structural changes occur in the smallest resistance
arteries. An increase in W/L of small but not of intermediate-size
resistance arteries was found in rats treated with 50 ng ·
kg-1 · min-1 Ang
II for 12 weeks and in rats treated with 100 ng ·
kg-1 · min-1 Ang
II for 4 weeks. The differential segmental arterial effects
of Ang II that we have observed in this and previous studies suggest
that Ang II exerts a direct trophic effect on small resistance
arteries. There is ample in vitro and some in vivo evidence that Ang II
is a trophic factor of vascular muscle. In vivo, it has been difficult
to separate the arterial pressure-dependent from the
pressure-independent effects of Ang II. In familial chronic diarrhea
and in Bartter's syndrome (clinical conditions of high plasma renin
activity), vascular hypertrophy develops in the absence of
hypertension.19 20 In rats treated with pressor
doses of Ang II, antihypertensive therapy prevented the hypertension
but not the development of vascular
hypertrophy.21 We have provided
evidence for stimulation of the vascular Na-K pump, a requirement for
growth,13 and increased protein and
glycosaminoglycan synthesis of vascular muscle in
rats receiving subpressor doses of Ang II.14
Interestingly, in the present study, there was also a trend for
increased W/L of small resistance arteries in salt-fed rats. This was
achieved with moderate salt supplementation that had no effect on the
BP of rats. In past studies, a large amount of salt was fed to rats for
5 to 8 months to produce hypertension and, in some rats, necrotizing
arteritis.22 Because of the large amount of
obligatory sodium intake to which these rats were exposed, the
relevance of these experiments to human hypertension is difficult to
assess. Another interesting aspect of salt supplementation in our study
is that the rats were evenly divided between responders (that is, those
that displayed structural vascular changes) and nonresponders. These
findings suggest a new definition of salt sensitivity and resistance;
rats that respond with an increase in W/L of small resistance arteries
to salt supplementation may be termed salt-sensitive and those that do
not, salt-resistant. The direct trophic effect of Ang II and,
in some rats, of dietary salt supplementation may form the basis of
synergism that we found between these 2 stimuli in producing
hypertension and structural vascular changes. Dietary sodium
supplementation may exert a trophic vascular effect through interaction
with cation-binding proteoglycans in the extracellular matrix. By
binding and attracting sodium ions, paracellularly localized
proteoglycans contribute importantly to transmembrane sodium gradient
and may thus regulate the magnitude of vasoconstrictor and trophic
vascular responses to agonists whose mode of action includes the
stimulation of sodium influx.23 Ang II is the
most important such agonist.23 Basement membrane
proteoglycans also serve as receptors for growth
factors.24 25 The binding characteristics of
these receptors are determined by the cationic milieu of the
interstitium, and depending on their molecular organization they may
either promote or inhibit growth. We have shown that pretreatment of
rats with high sodium diet potentiates Ang II–induced synthesis of
vascular proteoglycans, suggesting upregulation of Ang II
receptors.26 Finally, the basis for the synergism
between the pressor and vascular trophic effects of Ang II and salt may
be stimulation of vascular endothelin production, which has
vasoconstrictor and trophic effects of its
own.27
Besides dose dependence, there was also time dependence of the
development of structural vascular changes in rats. One half of an
effective dose of Ang II produced the same increase in W/L of
resistance arteries as the full dose when the time of administration
was increased three-fold. Whether this time dependence is due to the
cumulative effect of increases in BP or to a cumulative trophic action
of Ang II cannot be determined from these data. The concept that a
small, initially subpressor stimulus applied for a long period of time
may lead to structural vascular changes and hypertension was first
promulgated by Lever.9 The present study
provides additional data in support of this hypothesis. Time is
certainly a factor in the development of human hypertension. Chronic
hypertension is frequently ushered in by years of borderline
hypertension. Essential hypertension typically becomes established in
the third and fourth decades of life. During the developmental stage of
hypertension, the pressor stimulus may be so small as to be virtually
undetectable in comparison with that in a normotensive group of
subjects. The administration of Ang II in doses that are even smaller
than in the present study (such as the 25 ng ·
kg-1 · min-1 SC
that is expected, based on our measurements, to raise plasma Ang II
level by about 30%) for a long period of time promises to be a useful
model for the development of human essential hypertension.
The nature and development of structural vascular changes have been the
focus of numerous investigations into the pathogenesis of hypertension
because without these changes chronic hypertension does not
occur.4 5 6 7 Increased W/L of arteries is the
predominant lesion; it may result from increased vessel wall area
(hypertrophy or hyperplasia of vascular muscle) or from
reduced lumen due to restructuring of elements of the vessel wall
(remodeling).4 6 In genetic forms of experimental
and human hypertension, the primary pathological alteration appears to
be remodeling.4 In contrast, in the nongenetic
models of hypertension, medial hypertrophy seems to
predominate.4 7 In the only study (beside our
own) in which structural vascular changes have been investigated in an
Ang II–induced model of hypertension, the increase in W/L of large
mesenteric arteries was due almost entirely to cellular
hypertrophy.21 The present study
confirms this finding. In the same-size arteries that were investigated
by Griffin et al,21 we found vascular smooth
muscle cell hypertrophy without evidence for hyperplasia.
The importance of the present study lies in the analysis of
the relationship that exists between BP and structural vascular changes
in this model of hypertension. When BP load over time and W/L of small
resistance arteries of the various treated groups of rats were plotted
on the same graph, a clear dissociation between the 2
parameters was observed in the earliest stages of the
hypertensive process. It appears that this is the time to investigate
the relationship between pressure and structure in hypertension. The
dissociation between BP load and vascular structure in the earliest
stages of hypertension suggests that a direct trophic effect of Ang II,
in addition to its pressor one, is contributing to the development of
structural vascular changes. Further meticulous studies in the early
stages of hypertension are needed to confirm these observations.
In summary, the development of structural vascular changes in Ang
II–treated rats is dose- and time-dependent. The earliest changes are
detected in small resistance arteries and in arterioles. The
dissociation between BP load and W/L of mesenteric resistance arteries
in the earliest stages of hypertension suggests that direct trophic
stimulation of vascular wall by Ang II contributes to the development
of structural vascular changes. Dietary sodium supplementation
potentiates the trophic vascular and pressor effect of Ang II. The
administration of small, initially subpressor doses of Ang II to rats
for a long period of time promises to be a useful model for the
development of human essential hypertension.
Received May 26, 1998;
first decision June 11, 1998;
accepted June 18, 1998.
2.
Simon G, Abraham G, Cserep G. Pressor and subpressor
angiotensin II administration: two experimental models of
hypertension. Am J Hypertens. 1995;8:645–650.[Medline]
[Order article via Infotrieve]
3.
Simon G, Cserep G, Limas C. Development of structural
vascular changes with subpressor angiotensin II
administration in rats. Am J Hypertens. 1995;8:67–73.[Medline]
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4.
Mulvany MJ, Aalkjaer C. Structure and function of
small arteries. Physiol Rev. 1990;70:921–961.
5.
Korner PI, Angus JA. Structural determinants of
vascular resistance properties. J Vasc Res. 1992;29:293–312.[Medline]
[Order article via Infotrieve]
6.
Heagerty AM, Aalkjaer C, Bund SJ, Korsgaard N, Mulvany
MJ. Small artery structure in hypertension: dual processes of
remodeling and growth. Hypertension. 1993;21:391–397.
7.
Schiffrin EL. Vascular structure in
N-nitro-L-arginine methyl ester– induced
hypertension: methodological considerations for studies of small
arteries in hypertension. J Hypertens. 1995;13:817–821.[Medline]
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8.
Yamori Y, Igawa T, Tagami M, Kanbe T, Nara Y, Kihara
M, Horie R. Humoral trophic influence on cardiovascular
structural changes in hypertension. Hypertension.
1984;6(suppl III):III-27–III-32.
9.
Lever AF. Slow pressor mechanisms in hypertension: a
role for hypertrophy of resistance vessels. J
Hypertens. 1986;4:515–524.[Medline]
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10.
Csiky B, Simon G. Synergistic vascular effects of
dietary sodium supplementation and angiotensin II
administration. Am J Physiol. 1997;273:H1275–H1282.
11.
Harrap SB, Van der Merwe WM, Griffin SA, Macpherson F,
Lever AF. Brief angiotensin-converting enzyme
inhibitor treatment in young spontaneously hypertensive
rats reduces blood pressure long-term. Hypertension. 1990;16:603–614.
12.
Folkow B. Critical review of studies on salt and
hypertension. Clin Exp Hypertens. 1992;A14:1–14.
13.
Simon G. Stimulation of vascular Na-K pump with
subpressor angiotensin II in rats. Proc Soc Exp Biol
Med. 1992;199:424–431.[Medline]
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14.
Simon G, Altman S. Subpressor angiotensin
II is a bifunctional growth factor of vascular muscle in rats.
J Hypertens. 1992;10:1165–1171.[Medline]
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15.
Li T, Zimmerman BG. ß-Adrenergic–induced local
angiotensin generation in the rabbit hind limb is dependent
on the kidney. Hypertension. 1991;17:1010–1017.
16.
Abraham G, Simon G. Autopotentiation of pressor
responses by subpressor angiotensin II in rats.
Am J Hypertens. 1994;7:265–275.
17.
Csiky B, Simon G. Effect of neonatal
sympathectomy on the development of
angiotensin II-induced hypertension. Am J
Physiol. 1997;272:H648–H656.
18.
Simon G, Csiky B. Effect of neonatal
sympathectomy on the development of structural vascular
changes in angiotensin II-treated rats. J
Hypertens. 1998;16:77–84.[Medline]
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19.
Pasternak A, Perheentupa J. Hypertensive
angiopathy in familial chloride diarrhea. Lancet. 1966;2:1047–1049.[Medline]
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Sekiguchi S. Pathogenesis of Bartter's syndrome: functional and
histologic studies. Am J Med. 1977;63:467–474.[Medline]
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21.
Griffin SA, Brown WCB, McPherson F, McGrath JC, Wilson
VG, Korsgaard N, Mulvany MJ, Lever AF. Angiotensin II
causes vascular hypertrophy in part by a non-pressor
mechanism. Hypertension. 1991;17:626–635.
22.
Koletsky S. Pathogenesis of experimental hypertension
induced by salt. Am J Cardiol. 1961;8:576–581.
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© 1998 American Heart Association, Inc.
Scientific Contributions
Structural Vascular Changes in Hypertension
Role of Angiotensin II, Dietary Sodium Supplementation, Blood Pressure, and Time
Abstract
Top
Abstract
Introduction
Methods
Results
Discussion
References
Abstract—The dose and time
dependence of angiotensin II (Ang II)–induced hypertension
and structural vascular changes and the effect of dietary sodium
supplementation on these relationships were investigated. Male
Sprague-Dawley rats were treated with 50, 100, or 200 ng ·
kg-1 · min-1 Ang II subcutaneously for
4 or 12 weeks on normal sodium diet (0.7% NaCl) or with 50 ng ·
kg-1 · min-1 Ang II SC for 12 weeks on
high sodium diet (2% NaCl). Additional rats were sham-operated and fed
normal sodium (control rats) or high sodium diet. Plasma Ang II level
of rats receiving 100 ng · kg-1 ·
min-1 Ang II for 4 weeks was 26±5 pg/mL (mean±SEM, n=7)
compared with 11±2 pg/mL (n=15) in control rats
(P<0.03). Lumen and external diameters of small (50 to
100 µm OD) and intermediate-size (100 to 150 µm OD)
resistance arteries were measured in maximally dilated, pump-perfused
(55 to 60 mm Hg), in situ fixed mesenteric vascular beds of rats,
and wall-to-lumen ratios (W/L) were calculated. Large mesenteric
arteries of rats treated with 100 ng · kg-1
· min-1 Ang II for 12 weeks were examined to distinguish
hypertrophy from hyperplasia of vascular muscle. Tail
systolic blood pressure (BP) and W/L of resistance arteries of
Ang II–treated rats increased in a dose-dependent manner. Treatment
with 50 ng · kg-1 · min-1 Ang
II for 12 weeks had no significant effect on BP but produced the same
increase in W/L (+10%, n=8, P<0.06) as 100 ng ·
kg-1 · min-1 Ang II for 4 weeks (+9%,
n=18, P<0.05) (time dependence). A 2% NaCl diet for 12
weeks had no significant effect on either BP or W/L, but in combination
with 50 ng · kg-1 · min-1 Ang
II, it increased systolic BP by 31 mm Hg
(P<0.01) and W/L of small resistance arteries by 28%
(P<0.01) (synergism). In rats treated with 100 ng
· kg-1 · min-1 Ang II for 12 weeks,
arterial smooth muscle cell thickness was increased without
a change in the number of cell layers (hypertrophy). There
was a dissociation between the average BP load (the area under the
weekly systolic BP curve) of Ang II–treated rats and the W/L
of their mesenteric resistance arteries. Ang II–induced hypertension
and structural vascular changes are dose- and time-dependent and
synergistically enhanced by dietary sodium supplementation.
Dissociation between BP and vascular structure in Ang II–treated rats
suggests that a direct trophic effect of Ang II may contribute to the
development of structural vascular changes.
Key Words: arteries • morphometry • mesenteric circulation
Introduction
Top
Abstract
Introduction
Methods
Results
Discussion
References
The long-term administration of initially subpressor
doses of angiotensin II (Ang II) to rats mimics the
development of human renovascular and high-renin (Ang II) essential
hypertension.1 2 The onset of hypertension is
preceded by a prehypertensive period characterized by trophic
stimulation of vascular muscle and potentiation of pressor and
vasoconstrictor responses to Ang II itself (autopotentiation). This is
followed by the onset of hypertension and the development of structural
vascular changes. Preliminary data in our laboratory indicated that
this orderly process is both dose- and
time-dependent.1 2 3 By time dependence we mean
the phenomenon whereby one half of an effective dose of Ang II may take
twice as long as the full dose or longer to produce hypertension and
structural vascular changes. This is an important concept because it
suggests that a stimulus that is undetectable at any one point in time,
when applied long enough, may lead to hypertension. The first aim of
this investigation was to provide experimental data for the dose and
time dependence of Ang II–induced hypertension.
Methods
Top
Abstract
Introduction
Methods
Results
Discussion
References
Design of Experiments
Pathogen-free male Sprague-Dawley rats (Sasco, Omaha, Neb) on a
normal sodium (0.7% NaCl) diet were used throughout these studies.
Male rats were investigated to eliminate the changes in the
renin-angiotensin system that accompany the estrous cycle
in female rats. At the beginning of the experiments, the rats weighed
400 to 450 g. Young adult rats were chosen because Ang II
treatment may influence the growth of rats independently of its pressor
effect.11 Ang II was administered to rats in
three different doses (50, 100, and 200 ng ·
kg-1 · min-1) for
4 or 12 weeks to test the hypothesis that the agonist exerts a dose-
and time-dependent effect on the development of hypertension and of
structural vascular changes. Additional rats received high sodium (2%
NaCl) diet alone or in combination with 50 ng ·
kg-1 · min-1 Ang
II for 12 weeks to test for a synergistic effect of the 2 stimuli. It
has been estimated that 2% NaCl diet of rats corresponds to daily
intake of 
15 g of salt by humans,12 a salt
load that sometimes is encountered in clinical practice. A 2% NaCl
diet by itself was not expected to produce hypertension in
Sprague-Dawley rats. Sham-operated rats on normal sodium diet for 4 or
12 weeks served as controls for all the treatment groups, including the
rats on high sodium diet.
Alza model 2 ML4 (28-day) minipumps implanted
subcutaneously were used to deliver Ang II for 4 weeks. To deliver Ang
II for 12 weeks, the empty minipumps were surgically removed at weeks 4
and 8, and new ones were implanted. Procedures for the filling and
implantation of the minipumps have been previously
reported.13 The dose of Ang II
(Asp-Arg-Val-Tyr-Ile-His-Pro-Phe; Sigma Chemical Co) was 50, 100, or
200 ng · kg-1 ·
min-1 SC based on the body weight of rats at the
time of the implantation of the first minipump. Control rats and rats
on high sodium diet were fitted with empty resterilized minipumps to
reduce costs. The rate of delivery of the 4-week minipump is 2.5
µL/h; in previous experiments, we found that the delivery of this
volume of vehicle (0.01 N acetic acid) to control rats had no
detectable metabolic or hemodynamic
effect.13 14 The rats had free access to tap
water and received either normal (0.7% NaCl) or
sodium-supplemented (2% NaCl) diet. The diets were matched
for other ingredients, including potassium (200 mmol/kg). The
diets were prepared on order by Harlan Teklad.
Systolic BP was measured in restrained awake rats by the
tail-cuff method (Narco Biosystems) between 8 and 11 AM.
Measurements were made on at least 2 occasions before the insertion of
the first minipump to rule out spontaneous hypertension (SBP >130
mm Hg). Rats whose SBP exceeded 130 mm Hg were rejected from the
study. After insertion of the first minipump, the SBP of rats was
measured weekly for 4 weeks and then every 2 weeks for the rest of the
experiment. The BP load that rats were exposed to during the
experiments was calculated as the area under the SBP curve. The area
was calculated by subdividing the BP curve of each rat into weekly or
biweekly trapezoids, by computing the area of each trapezoid, and,
finally, by adding up the calculated areas. The rats were weighed to
the nearest 1 g on the day of the final experiments.
For direct measurement of BP, rats were fitted with a femoral
artery catheter 7 to 10 days before the end of the 4- or 12-week
treatment period. The operation was performed in pentobarbital (45
mg/kg IP)–anesthetized rats. Through a skin incision in the
left groin, a polyvinyl chloride catheter was advanced into the
abdominal aorta through the femoral artery and fixed in place. The
distal end of the catheter was tunneled subcutaneously with the aid of
a trocar from the groin to the back of the neck, where it was
exteriorized through a puncture wound. Postoperatively, the rats
received acetaminophen for analgesia in their drinking
water (1.5 mg/mL) for 1 day. After a 3-day recovery period, the
arterial catheter was connected to a pressure transducer
(Kent Scientific Corp), and the mean arterial BP (MAP) was
monitored continuously for 2 hours between 8 AM and 12
PM on 3 separate days. During monitoring, rats moved freely
in their boxes. The filtered analog signals were digitized with a
Maclab analog/digital converter and sampled using an Apple computer
with system 7 software. The average MAP was calculated from collected
data for each day, and the average daily MAP during 3 days of
measurement was calculated.
The techniques used for the in situ fixation of the mesenteric
and left renal vascular bed of rats have been previously
reported.3 Briefly, the rats were
anesthetized with 75 mg/kg chloralose IV and allowed to breathe
spontaneously through a tracheostomy tube. After systemic
anticoagulation (1000 U IV) and vasodilatation (500 µg papaverine IV
per rat), the mesenteric and left renal circulation of rats was
pump-perfused retrogradely through the abdominal aorta with aerated
(5% CO2, 95% O2)
Krebs-Ringer bicarbonate solution at 37°C, to which 75 µg/mL
papaverine was added. The inferior vena cava was cut open
to allow free circulation of the perfusate through the left
kidney and the mesentery. Perfusion pressure in the aorta was adjusted
to 55 to 60 mm Hg and kept constant by adjusting the pump flow
rate for the rest of the infusion period. The pressure range of 55 to
60 mm Hg was chosen because we have found previously that after
maximal systemic vasodilatation, MAP falls to this level in rats
treated with Ang II and in control rats.16 The
mesenteric and left renal vascular beds were perfused sequentially with
Krebs-Ringer solution for 8 to 10 minutes, with fixative containing
2.5% glutaraldehyde, 1.86% sucrose, and 0.063 mol/L
phosphate buffer (pH 7.4, 400 mOsm) for 15 minutes, and with 0.200
mol/L phosphate buffer for 8 to 10 minutes. At the end of perfusion,
the left kidney and the entire small intestine and mesenteric vascular
arcade were removed for further processing.
For the purposes of this study, morphometric measurements were
restricted to mesenteric arteries. The small intestine was cut into
2- to 3-cm segments, each segment retaining its own vascular
arcade, and post-fixed in 10% buffered formalin. A wooden stick was
inserted into the lumen of the intestinal segments to straighten the
junction of the intestinal wall and the vascular arcade where the cuts
for histological sections were going to be made. Three
to four intestinal segments were dehydrated through graded series of
ethanol and were infiltrated with and embedded in paraffin. Cuts were
made at a right angle and parallel to the mesenteric intestinal
junction; this resulted in cross-sectional cuts of small mesenteric
arteries. Sections of 4 µm thickness were stained with
hematoxylin and eosin, which delineated the outer media and adventitia.
Two criteria were used to select arteries for morphometric
measurements. First, by inspection, the thickness of the vessel wall
had to be uniform throughout its circumference. Second, only vessels
with a long-to-short axis ratio of <1.50 were measured. In the
present study, 2 categories of resistance arteries, 1 with external
diameter of 50 to 100 µm (small) and the other with external
diameter of 100 to 150 µm (intermediate-size), were
investigated. Morphometric measurements of external and lumen diameters
along the long and short axes of cross-sectionally cut arteries were
obtained under x450 magnification with a calibrated filar
micrometer. Measurement of external diameter extended from
the margin of the outer media to the margin of the outer media of the
opposing wall. The 2 measurements of external and lumen diameters were
averaged. The observer (G.S.) was blinded as to the treatment that the
rats received. Measurements were made on a minimum of 3
cross-sectionally cut arteries in each of the 2 vessel categories
investigated (see above). Wall thickness and W/L and the mean of each
parameter were calculated for the 2 categories of vessels
for each rat.
Results are presented as mean±SEM. Repeated-measures
ANOVA (1 factor within, 1 between; Superanova, ABACUS Concepts) was
used to compare weekly BPs of Ang II–treated rats and of
sodium-supplemented rats with those of control rats. Linear regression
analysis was used to correlate the average weekly tail SBP and
the average MAP of catheterized rats.
Results
Top
Abstract
Introduction
Methods
Results
Discussion
References
By inspection, all rats remained healthy to the end of the study.
The body weight of rats undergoing 4 weeks of treatment was as follows:
controls, 497±25 g (n=8); and rats treated with 50, 100, and 200
ng · kg-1 ·
min-1 Ang II, 467±18 (n=6), 468±7 (n=22), and
474±9 (n=8) g, respectively. In the 12-week treatment groups, the body
weight was as follows: controls, 514±24 g (n=7); rats treated with 50
and 100 ng · kg-1 ·
min-1 Ang II, 543±26 (n=8) and 598±17 g (n=9,
P<0.01 compared with controls), respectively; rats treated
with 50 ng · kg-1 ·
min-1 Ang II and fed 2% NaCl, 467±27 g (n=6);
and rats fed 2% NaCl, 520±22 g (n=17). The tail SBP of rats is shown
in Figures 1
and 2. There was rapid development of severe
hypertension in rats receiving 200 ng ·
kg-1 · min-1 Ang
II SC for 4 weeks. Because of the severe hypertension that developed in
these rats, 12-week treatment of rats with this dose of Ang II was not
attempted. The development of hypertension was more gradual in rats
treated with the 100 ng · kg-1 ·
min-1 dose of Ang II, reaching moderate levels
by 4 weeks of treatment. In rats treated with 50 ng ·
kg-1 · min-1 Ang
II, BP rose transiently at 3 and 4 weeks but returned to baseline by
the end of the 12-week treatment period (Figures 1 and 2). (This may
have been due to weight gain of rats that resulted in a diminishing
dose of Ang II per kilogram of body weight as the experiments
progressed.) During the 12-week treatment, the BP of rats treated with
50 ng · kg-1 ·
min-1 was not different from that of control
rats. The BP curve of control rats and of rats receiving the 2% NaCl
diet overlapped during the entire treatment period. However, when the
2% NaCl diet was combined with the administration of 50 ng ·
kg-1 · min-1 Ang
II, SBP of rats began to rise by week 3, reaching mildly elevated
levels by week 12. At the end of 4 weeks of treatment, the plasma Ang
II level of rats receiving 100 ng ·
kg-1 · min-1 Ang
II SC (26±5 pg/mL, n=7) was increased compared with that of control
rats (11±2 pg/mL, n=15) (P<0.03).
View larger version (15K):
[in a new window]
Figure 1. Tail SBP (mean±SEM) of rats treated with 50, 100,
or 200 ng · kg-1 · min-1 Ang II
SC for 4 weeks and of control rats. The BP curve of Ang II–treated
rats was compared with that of control rats by repeated-measures ANOVA.
B1 and B2 indicate 1st and 2nd baseline BP; arrowhead indicates start
of treatments.
View larger version (17K):
[in a new window]
Figure 2. Tail SBP (mean±SEM) of rats treated with 50 or
100 ng · kg-1 · min-1 Ang II
SC, 2% NaCl diet, or 2% NaCl diet+50 ng ·
kg-1 · min-1 Ang II SC for 12 weeks
and of control rats. The BP curve of treated rats, including the rats
on 2% NaCl diet, was compared with that of control rats by
repeated-measures ANOVA. For details, see Figure 1 
. and 4. A
dose-dependent increase in the W/L of both categories of vessels was
observed, but the increases were generally greater in small arteries.
The increase in the W/L of small arteries of rats treated with 50
ng · kg-1 ·
min-1 Ang II for 12 weeks was of borderline
statistical significance (P<0.06) (Figure 3). The effect of
dose and time of administration of Ang II on W/L was analyzed
by 2-factor ANOVA (see above). In the small arteries, there was a
significant independent effect of dose of Ang II (P<0.01)
but not of duration of treatment (P<0.20) on wall
thickening (Figure 3). In intermediate-size arteries, the effect of
both dose and duration of treatment was statistically significant
(P<0.02 and P<0.01, respectively) (Figure 4).
View larger version (29K):
[in a new window]
Figure 3. Wall-to-lumen ratio (mean±SEM) of small
mesenteric resistance arteries (50 to 100 µm OD) of rats on the
various 4-week (solid bars) and 12-week (hatched bars) treatment
regimens; number of rats is shown at the base of each bar. W/L of Ang
II–treated and 2% NaCl-fed rats was compared with that of appropriate
control rats by 2-factor ANOVA. *P<0.05;
P<0.01; 
P<0.001.
View larger version (25K):
[in a new window]
Figure 4. Wall-to-lumen ratio (mean±SEM) of
intermediate-size mesenteric resistance arteries (100 to 150 µm
OD) of rats on the various 4-week (solid bars) and 12-week (hatched
bars) treatment regimens; number of rats is shown at the base of each
bar. For statistical analysis of results, see Figure 3 .
*P<0.01; P<0.05;
P<0.02.. For reasons stated in Methods,
statistical comparison of small and intermediate-size artery dimensions
in Ang II–treated and control rats was confined to that of W/Ls. W/L
of both categories of arteries was increased in Ang II–treated rats.
Statistical comparison of large-artery dimensions in the 2 groups
revealed increased wall thickness, W/L, and vascular smooth muscle cell
thickness in Ang II–treated rats without a change in the number of
smooth muscle cell layers. This finding indicates vascular muscle
hypertrophy rather than hyperplasia. Arterial
wall hypertrophy in Ang II–treated rats is illustrated in
Figure 5.
View this table:
[in a new window]
Table 1. Morphometry of Mesenteric Arteries of Rats Treated With
100 ng · kg-1 · min-1 Ang II SC
for 12 Weeks and of Control
Rats
View larger version (109K):
[in a new window]
Figure 5. Light micrograph of 2nd-order mesenteric artery
from a rat treated with 100 ng · kg-1 ·
min-1 Ang II SC for 12 weeks (left panel) showing
thickened media due to smooth muscle hypertrophy compared
with 2nd-order artery from a sham-operated normotensive control rat
(right panel). Toluidine blue–stained semithin sections, x1000
original magnification. ). This finding indicated that the
latter was an acceptable measure of the BP load of rats. This permitted
us to construct the final summary figure (Figure 7) on which the W/Ls of small arteries of
the various treatment groups are plotted against their average BP load,
calculated as the area under the SBP curves of rats (Figures 1 and 2)
(see Methods). Included in Figure 7 are the results from a previous
study in which neonatally sympathectomized adult male rats were treated
with 200 ng · kg-1 ·
min-1 Ang II SC for 4 weeks or sham-treated
(control).17 The figure illustrates that there is
a dissociation between the BP load that these rats were exposed to and
the W/L of their small mesenteric arteries, especially in the earliest
stages of hypertension. This impression was confirmed by linear
regression analysis of the BP load and W/L of small mesenteric
arteries of individual rats that were treated with Ang II for 4 weeks
(r=0.15, n=32, NS) or for 12 weeks (r=0.04,
n=23). Rats fed a 2% NaCl for 12 weeks were not included in this
analysis. A similar dissociation was found between the BP load
of rats and the W/L of their intermediate-size mesenteric arteries
(data not shown).
View larger version (25K):
[in a new window]
Figure 6. Correlation of intra-arterial and tail
SBP of rats treated with 50 ( 
) or 100 ng ·
kg-1 · min-1 Ang II SC (
) for 4
weeks or with 50 ng · kg-1 ·
min-1 Ang II SC for 12 weeks (
) and of control rats
(
).
View larger version (16K):
[in a new window]
Figure 7. Graphic plot of the average wall-to-lumen ratio
(mean±SEM) of small mesenteric resistance arteries (50 to 100
µm OD) of the various treated groups of rats against their BP load
(the area under the weekly SBP curve of rats) (mean±SEM) compared with
that of control rats. and indicate 4- and 12-week treatment,
respectively; 50, 100, and 200 indicate the dose of Ang II in ng
· kg-1 · min-1 SC. Included are
sympathectomized control rats (Sx) and sympathectomized rats treated
with 200 ng · kg-1 · min-1 Ang
II SC (Sx+A II 200) from a previous study.17 For
statistical analysis of the effect of dose and time of
administration of Ang II on W/L, see Figure 3 and Results. AII
indicates angiotensin II.
Discussion
Top
Abstract
Introduction
Methods
Results
Discussion
References
The findings of this study provide evidence for dose and time
dependence of Ang II–induced hypertension and of the concomitant
development of structural vascular changes. A lesser dose of Ang II may
lead to the same increase in W/L of resistance arteries as a higher
dose as long as the period of treatment is increased. Subthreshold or
near-threshold stimuli, such as 2% NaCl diet and 50 ng ·
kg-1 · min-1 Ang
II SC, applied simultaneously lead to hypertension and the
development of structural vascular changes, demonstrating true
synergism. When the W/L of resistance arteries and the BP load over
time of several groups of Ang II–treated rats were plotted together on
the same graph, a clear dissociation between structural vascular
changes and BP was demonstrated, especially in the earliest stages of
hypertension.
Acknowledgments
This work was supported by research funds from the Department of
Veterans Affairs. The authors thank Dr Ben Zimmerman, Department of
Pharmacology, University of Minnesota, for measurements of plasma Ang
II concentrations and Dr John W. Osborn, Department of Veterinary
Biology and Neuroscience Graduate Program, for the demonstration of
long-term intra-arterial catheterization of
rats. Steve Altman and Paul Talarico provided skillful technical
assistance.
Footnotes
Dr Illyes is currently at the 2nd Department of Pathology, Semmelweis Medical University, Budapest; Dr Csiky is currently at the 2nd Department of Medicine, University Medical School of Pecs, Pecs, Hungary.
References
Top
Abstract
Introduction
Methods
Results
Discussion
References
1.
Simon G, Abraham G. Angiotensin II
administration as an experimental model of hypertension. In: Laragh JH,
Brenner BM. Hypertension: Pathophysiology, Diagnosis, and
Management. 2nd ed. New York, NY: Raven Press Publishers;
1995:1423–1435.
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