(Circulation. 1995;91:1161-1174.)
© 1995 American Heart Association, Inc.
Articles |
Hyperhomocysteinemia-Induced Vascular Damage in the Minipig
Captopril-Hydrochlorothiazide Combination Prevents Elastic Alterations
From INSERM and Laboratory of Biological Chemistry, School of Pharmacy (P.H.R., A.F., A.B., V.L., M.M.F., P.C., H.B., O.G., R.C., D.G.), and Departments of Vascular Surgery (P.P.) and Cardiology B (R.L.), Hospital La Timone, Marseilles, and Rhône-Poulenc-Rorer/Theraplix Laboratory (J.G.), Paris, France.
Correspondence to Pierre H. Rolland, INSERM, Laboratoire de Chimie Biologique, Faculté de Pharmacie, 27 Bd Jean-Moulin, 13385 Marseille CEDEX 5, France.
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Abstract |
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 Top Abstract IntroductionMethods Results Discussion References |
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Background Previous attempts in animals failed to reproduce the metabolic, pathological, and clinical situations encountered in homocystinuric patients. Minipigs on a methionine-rich caseinate-based diet, however, have a special long-lasting postprandial plasma accumulation of methionine, the metabolic precursor of homocysteine. We hypothesized that these minipigs develop hyperhomocysteinemia in the long term. Angiotensin-converting enzyme (ACE) inhibition prevents atherogenic alteration of viscoelastic functions of arterial pulsatility and compliance and reduces fragmentation of vascular elastic laminae in the minipigs. We consequently analyzed the therapeutic effects of the captopril-hydrochlorothiazide combination against the typical hyperhomocysteinemia-induced alterations of vascular elastic features.
Methods and Results Thirty-two Götingen minipigs were randomized as control diet-fed (C), captopril (25 mg/d)/hydrochlorothiazide (12.5 mg/d)-treated C (C+Cp), caseinate-based diet-fed (M), and M+Cp minipigs. After 4 months, M and M+Cp animals had hyperhomocysteinemia (9.64±4.10 µmol/L, n=16) compared with C and C+Cp minipigs (5.67±1.14 µmol/L, n=16) (P<.05). In the M group, one minipig died from thromboembolic syndrome, and one had pulmonary infarction. M minipigs presented with systolic-diastolic hypertension and extended reactive hyperemia, as well as a mega-artery syndrome in hyperpulsatile arteries due to expanded volumetric compliance, curtailed stiffness, strengthened vascular tension, and prevalence of the viscous wall component. In their arterial tree, hypertrophic endothelial cells covered a thickened subendothelial space. Major elastic lamina dislocations were observed, as well as hypertrophy and reorientation of smooth muscle cells, resulting in the settlement of spreading pathways for medial cells between muscular laminae. In C+Cp and M+Cp animals, serum and lung ACE activity were inhibited by 74% and 40%, respectively. Although the treatment with captopril-hydrochlorothiazide did not modify the hyperhomocysteinemia per se, the therapeutic effects of the drug combination are made evident by the absence of death and ischemic diseases in the M+Cp group. Specifically, the drug combination prevented diastolic hypertension and improved aortic blood flow by normalizing peripheral resistances, abolished the vascular hyperpulsatile characters, and restrained the fragmentation and the splitting of elastic fibers in capacitance arteries. In contrast, the drugs slightly prevented systolic and mean hypertension. In addition, the aortic stiffness and stress response remained altered and vascular smooth muscle cell hypertrophy was still observed in the M+Cp group.
Conclusions In minipigs, the present methionine-rich caseinate-based diet induced hyperhomocysteinemia, which reproduces the metabolic and histopathological situation found in homocysteic patients. Our results show that hyperhomocysteinemia-induced vascular alterations favor the viscous component of the wall rheology to the detriment of the elastic component. Furthermore, they extend to hyperhomocysteinemia the therapeutic effects characteristically shared by ACE inhibitors in association with hydrochlorothiazide against the atherogenic activation of elastinolytic processes.
Key Words: hemodynamics • rheology • angiotensin • diuretics • minipigs
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Introduction |
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In patients with homocystinuria, ie, the rare homozygous condition of cystathionine ß-synthase deficiency, the most life-threatening symptom is premature atherosclerosis and thromboembolism.1 In addition, there is now evidence that mild homocysteinemia, which is related to the more common condition of heterozygosity, is an independent risk factor for occlusive vascular disease.2 3 4 5 Homocysteine and the metabolic intermediates of the methionine transsulfuration pathway are causal agents in the development of vascular disease in patients with hyperhomocysteinemia originating from different sources.1 6 However, the relation between homocysteine and vascular disease remains inconclusive because of the absence of relevant experimental models for hyperhomocysteinemia, despite previous attempts made to treat rabbits, baboons, and pigs with homocysteine thiolactone, methionine, or homocysteic acid or to feed them vitamin B6–deficient diets.7 8 9 10 11 12 13 14 15 16 17 18
The present studies originated from the hypothesis we made that hyperhomocysteinemia will develop in adult minipigs fed a dietary regimen whose protein content excludes purified methionine-rich calcium caseinates. A threefold increase in methionine postprandial plasma concentrations is noticed in the animals on the caseinate-based diet compared with the soy protein-based diet, and it was substantially higher than that of all the other plasma amino acids.19 It was therefore conceivable that such a hypermethioninemia led to hyperhomocysteinemia in the long term because of the overloading of the methionine transsulfuration pathway. We report here that significant hyperhomocysteinemia developed in minipigs fed purified L-methionine–rich calcium caseinates with no other associated serum metabolic disorder.
In humans, vitamin therapy is traditionally considered to normalize increased plasma homocysteine concentrations originating from metabolic defects in the enzymatic control of homocysteine metabolism as well as low levels of involved cofactors (ie, folate, vitamin B12, and vitamin B6).1 2 3 4 5 6 In contrast, the present experimental study involves a vascular therapeutic approach addressing the fibrous thickening of the intima,6 20 the fragmentation of the internal elastic lamina,6 20 21 and the fraying and splitting of muscle fibers and elastic fibers within the media in homocystinuric patients.20 Angiotensin- converting enzyme (ACE) inhibition improves large-artery compliance in hypertensive rats22 and humans23 and in atherosclerotic minipigs at a low therapeutic dose that can be used in the clinical settings.24 25 We recently demonstrated that the therapeutic effects of ACE inhibitors in atherosclerotic minipigs24 25 are due to their specific properties of preventing atherogenic alteration of viscoelastic functions of arterial pulsatility and compliance and reducing fragmentation of aortic elastic laminae.24 25 26 27 Since the combination of captopril plus hydrochlorothiazide improves aortic distensibility and viscoelasticity in a substantially more efficient manner than does captopril alone,28 29 it is worthwhile to investigate whether an ACE inhibitor associated with a thiazide diuretic prevents the hyperhomocysteinemia-induced alterations of arterial wall viscoelastic structures and functions.
The purposes of the present study were to define the functional and structural vascular consequences of hyperhomocysteinemia and to investigate the vascular therapeutic effects of the captopril-hydrochlorothiazide combination by noninvasive and invasive assessments of the arterial flow conditions in hind-limb arteries and by investigation of the histopathology of the arterial tree.
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Methods |
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Animals, Standard and Methionine-Rich Diets, and Captopril Plus Hydrochlorothiazide Treatment
The animals were handled in accordance with the INSERM U-278 Animal Care and Use Committee, which granted approval of this experimental protocol. Thirty-two male Götingen minipigs (Genipig SA) (10 months old; body weight, 20.9±1.2 kg) were housed together with natural daylight and free access to water for a 2-week acclimatization period. Minipigs then were randomized into four groups (n=8 per group): control diet (C), control diet and captopril-hydrochlorothiazide-treated (C+Cp), methionine-rich diet (M), and methionine-rich diet and captopril-hydrochlorothiazide-treated (M+Cp) minipigs. Control animals were given a standard diet (500 g, 1850 calories individually per day) consisting of cereals, fish and vegetable proteins and cellulose, vitamins, and mineral salts (21 g% proteins with 2 g% fish proteins, 65.5 g% carbohydrates, and 4 g% lipids; Breeding Diet UAR 127). The daily ration given to C and C+Cp animals contained 1.28 g methionine. Thus, a pig weighing 25 kg received methionine 50 mg · kg-1 · d-1, ie, the usual daily absorption dose of humans and pigs.30 31 The M and M+Cp animals were on a protein methionine-rich diet consisting of purified, delipidated calcium caseinates (30 g%), potato flour (58 g%), large bran and Wende cellulose (8 g%), fish starch (2 g%), and vitamins, salts, and hog nectar (2 g%), which contained 30 g% proteins, 62 g% carbohydrates, and 1.5 g% lipids. Calcium caseinates M220 (Unilait) consisted of proteins (90 g%), water (6.5 g%), calcium (1 g%), phosphorus (0.6 g%), sodium and potassium (0.1 g%), lactose (0.15 g%), and lipids (1.4 g% with cholesterol <0.4 g%). For 4 months, M and M+Cp animals were given a 400-g/d ration, corresponding to 2000 cal and 3.45 g methionine. No significant differences in calcium content existed between control and methionine-rich diet daily rations. The animals were individually fed the appropriate diet, and we checked the quantity for each animal. The animals underwent surgery as M and M+Cp matched pairs within 2 consecutive days and were interspersed with control animals.
Drug Administration, Blood Collection and Processing, Serum
Biochemistry, Assay of ACE Activity
In the present experimental
protocol, C+Cp and M+Cp animals,
independent of weight, were given constant 25-mg captopril and 12.5-mg
hydrochlorothiazide daily doses as a single tablet inserted in
their food. Control and M animals were similarly given a
placebo-matched tablet. We checked carefully that the minipigs
swallowed the tablets. Blood samples were drawn in the morning, ie, 24
hours after the last treatment and feeding, from the vena cava of pigs
in dorsal recumbency. Blood (10 mL) collected into nonanticoagulated
tubes stood for 2 hours at 37°C in a water bath before centrifugation
(2500 rpm, 15 minutes, 4°C). A further 10 mL of blood was collected
on K3-EDTA Vacutainers (Becton Dickinson Europe) before centrifugation.
Serum and plasma samples were either processed within 1 day or frozen
(-25°C) immediately. Serum metabolic parameters and ACE activity
were evaluated before and after 1, 2, and 3.5 months with appropriate
Boehringer Mannheim kits working with Hitachi 717 Systems (Boehringer
Mannheim Inc) as previously reported.26 27
Hippuryl-gly-gly reference peptide was obtained from Boehringer
Mannheim and 2,4,6-trinitrobenzene sulfonic 1 mol/L aqueous solution
from Fluka Chemie AG. Tissue ACE assay was performed similarly on lung
homogenates (1 g lung tissue was homogenized in 5 mL Krebs/Ringer's
lactate buffer, pH 7.4; sampling volume for ACE assay, 20 µL).
Although it is acknowledged that the pharmacological efficiency of ACE
inhibitor may vary from organ to organ, ACE activity was assayed in ACE
activity–rich lungs because we previously found that assay of ACE
activity in the pulmonary tissue was reproducible and much less subject
to interfering background than the arterial wall (P.H.R., unpublished
results; see Reference 32 for discussion). Reference plasma standards
for ACE activity (
150 U/mL) were from Boehringer Mannheim GmbH.
Plasma aminograms were determined after reduction of plasma samples by
dithiothreitol at pH 7.0 to release free homocysteine from its
conjugate forms (protein-bound homocysteine, homocystine, and
homocysteine-cysteine mixed disulfide).33 Reduced plasma
samples then were chromatographed on a cationic ion exchange resin in a
Beckman AA 6300 amino acid analyzer (Beckman Instruments, Inc) with
lithium citrate buffer as eluent solvent and ninhydrin as revealing
agent, as described elsewhere.34
Noninvasive Assessment of Hemodynamics, Surgical Procedure,
Invasive Measurement of Hemodynamics, Aortic Wall Rheology, and
Vascular Reactivity
Noninvasive assessment of hemodynamics and
vascular reactivity
was carried out by plethysmographic volume measurements of the caudal
artery with a pediatric sphygmomanometric cuff inserted around the
minipig's tail upstream of a plethysmographic mercury-in-Silastic
strain gauge35 (Plethysmograph, Echomed). Reactive
hyperemia was reproduced in the caudal vascular bed of pigs throughout
the restoration of blood flow in the caudal artery after a 2-minute
period of occlusion, and downstream ischemia was produced by inflating
the cuff to 50 mm Hg above systolic pressure. Plethysmographic results
are expressed as volts from the amplification of the signal made under
constant conditions of measurement and continuous recordings. Results
are average values from five consecutive cardiac cycles, of which the
median cycle had the maximal systolic volume value.
Surgery and
measurements of hind-limb hemodynamics and aortic wall
rheology were performed as previously
described24 25 36
from ultrasonic determination of pulsatile external aortic diameter and
pulsatile and mean aortic blood pressures and flows in the abdominal
aorta at equal distance between renal and lower mesenteric arteries.
Analog data were digitized (analog-digital converter AD 2821 F, A/D
Systems) at 8000 samples per second per channel over a period of 20
cardiac cycles and stored in computers (Hewlett-Packard Vectra, Digitex
386-SX). Heart rate was on-line computerized. Within one group of
animals, we determined individual-average values of hemodynamics and
rheological parameters, and we obtained population-average
graphic representation of relations between pulsatile flow,
pressure, and arterial wall diameter. Individual-average flow,
pressure, and diameter cycles, identical in duration, were directly
obtained from 20 native cycles whose synchronization was triggered by
the R-wave peak of the ECG. Individual hemodynamics and rheological
parameters were obtained as described below.
Graphic representations of population-average flow, pressure, and arterial wall diameter cycles were directly obtained from individual-average synchronized cycles after computerized normalization, as follows: the duration (in milliseconds) of each cycle was precisely determined, and each cycle was further divided into 500 equal segments whose durations were evaluated. Cycles were then normalized by giving their individual segment the segment duration of the longest cycle, thereby "stretching" the cycles to a common length. Population-average hysteresis loops representative of pressure-diameter or pressure-flow relations then were computerized from x-y plotting of instantaneous values of pressure and diameter or pressure and flow.24 36 37 The widths and slopes of the hysteresis loops obtained are suitable indicators of the viscous and elastic components of the viscoelastic properties of the arterial wall.24 36 37 In addition, x-y graphs of instantaneous pulsatile and mean aortic blood flows versus aortic blood pressure enable detection of changes in systolic and diastolic blood flow, which may be masked in mean values.24
From
mean, systolic, diastolic, and pulse aortic blood pressure and
flow and mean, external, and internal diameter, we evaluated the
hindquarter peripheral resistance, characteristic input impedance,
aortic volumetric compliance, aortic wall stiffness, Young's elastic
modulus (Ep), mean aortic wall thickness, and midwall radial aortic
stress (
), as previously described.24 37 It
should be
emphasized that Ep refers to the elastic stiffness of the aortic wall
per se and is not to be confused with the terms 
V/P or
P/D,
which refer to the compliance and stiffness, respectively, of the aorta
as a hollow structure.24 37
Histological Analysis of the Vasculature
Histological changes
were evaluated in the abdominal aorta
(in a 1.5-cm-long segment of abdominal aorta located at middistance
between the left renal artery and aortic trifurcation), in the left
interventricular coronary artery (LIVCA, in a 0.5-cm-long segment of
LIVCA sampled immediately after the circumflex artery bifurcation from
the left coronary trunk), and in the common carotid bifurcation (in a
0.5-cm-long segment of the distal left common carotid artery centered
on the internal and external carotid artery bifurcations), as
previously described.24 25 Immediately after the pigs
were
killed (20 mL KCl, 10% wt/vol IV), vessel segments were carefully
rinsed in ice-cold isotonic saline solution. The segments of aorta
(further sectioned transversely into four pieces) and coronary and
carotid arteries were fixed in Bouin's solution for 24 hours.
Longitudinal and transverse 4-µm serial sections were obtained and
stained alternately with hematoxylin, eosin, and safranin for general
observation, Masson's green trichrome for collagen and connective
tissue, and Darrow's orcein for specific staining of elastic tissue,
as previously described.24 38 All histological
analyses
were blinded.
Statistical Analysis
Data are reported as mean±SD. All
data from individual minipigs
were computed and stored on STATGRAPHIC software.
Statistical analysis was performed with Student's t
test, ANOVA, and a Mann-Whitney U test. A value of
P<.05 was considered statistically significant.
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Results |
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Effects of Diets and Drugs on Animal Welfare, Serum Metabolic Parameters, and Plasma and Pulmonary ACE Activity
Daily food intake was monitored and was not different for minipigs within the groups. All animals remained healthy throughout the treatment period and survived until euthanasia except two minipigs in the M group, which underwent major cardiovascular events at the time of the fourth monthly blood sampling for serum biochemistry assay (see animals 56 and 63 in Table 1
). The next day, one of
these animals had an acute cerebral stroke as encephalic artery
thrombosis and subsequent tetraplegia and died the day after due to
vena cava thrombosis and pulmonary embolism, as pathologically
documented at autopsy. The other pig showed no clinical signs of
pathological lesions and remained alive until surgery 20 days later,
when consolidated, massive infarctus in the left lung cephalic lobe was
documented. To prevent the introduction of bias into the protocol,
these animals remained free of therapeutics. The other minipigs in
the M and M+Cp groups tolerated the methionine-rich diet well and
gained as much weight as the controls fed standard diet. Final weights
were 29.3±9.8, 28.1±3.8, 34.1±4.6, and 33.6±3.4 kg
for C, C+Cp, M,
and M+Cp groups, respectively. Neither the diets nor the drugs induced
significant changes between groups in serum biochemistries (Table
1).
In contrast, the caseinate-based diet induced both
hypermethioninemia and hyperhomocysteinemia (Table 2). The mean
preprotocol plasma homocysteine level for
all the animals was 4.91±1.20 µmol/L (n=32). Significant
hyperhomocysteinemia was detectable after 1 month and stabilized after
2 months in the M and M+Cp groups (data not shown). After 4 months, the
mean plasma homocysteine for all the animals fed methionine-rich diet
was 9.64±4.10 µmol/L (n=16), and for all the animals fed
standard
diet, it was 5.67±1.14 µmol/L (n=16), which was not
significantly
different from preprotocol values. At that time, the animals in the M
and M+Cp groups tended to have increased plasma levels of
-aminobutyrate, the terminal metabolite of methionine in plasma.
Plasma aminograms provide evidence that no other excess or deficiency
in plasma amino acid contents was detectable among groups of
animals.
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Serum ACE activity remained constant in placebo-treated
animals
(C and M groups) throughout the duration of the protocol, whereas after
4 months, ACE activity was inhibited by 70% and 74% in animals
treated with captopril-hydrochlorothiazide in the M+Cp and C+Cp
groups,
respectively (Table 3). Plasma ACE activity was
inhibited by half within 1 month in treated animals (data not shown)
and decreased progressively until the end of the protocol. Compared
with untreated animals in the C and M groups, ACE inhibitor treatment
induced 62% and 38% inhibition of tissue ACE activity in lung
parenchyma of animals in the C+Cp and M+Cp groups, respectively
(Table 3). However, there was no difference in lung ACE
inhibition between
control and homocysteinemic minipigs because there was no significant
difference in pulmonary ACE activity between the C and M groups or
between the C+Cp and M+Cp groups.
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Noninvasive Hemodynamics and Vascular Reactivity
Four months
after the beginning of the protocol, the animals were
used to being kept in dorsal recumbency and to remaining quiet in this
position while being investigated for baseline values of noninvasive
hemodynamics and reactive hyperemia. Heart rate was found to be
significantly increased only in hyperhomocysteinemic animals compared
with other animals (P<.05), being 86±7, 88±8,
111±9, and
102±5 beats per minute in the C, C+Cp, M, and M+Cp groups,
respectively. Baseline values of systolic blood pressure, similarly,
were substantially higher in animals of the M group (178.1±22.6
mm Hg) compared with either the C (111±10.5 mm Hg), C+Cp
(123.1±8.0
mm Hg), or M+Cp (138.8±24.7 mm Hg) groups (P<.05).
There
were no significant differences in the basal measurements between the
M+Cp and both the C and C+Cp groups. Reactive hyperemia was observed
in
all the animals as markedly increased pulse blood volume from baseline
values after blood flow restoration. Typical reactive hyperemia is
illustrated in Fig 1 for animals in the C, M, and M+Cp
groups. There were no noticeable differences between animals in the C
and C+Cp groups. Plethysmographic pulse volumes during reactive
hyperemia were significantly increased only in M animals compared
with those of the C, C+CP, or M+Cp animals (P<.05),
being
1.00±0.60, 1.09±0.80, 2.94±0.80, and 1.35±0.81 V
in the C, C+CP, M,
and M+Cp groups, respectively. The increased pulse volume reflecting
the hyperpulsatile character of caudal vessels during reactive
hyperemia in animals of the M group was associated with a sustained
response to transitory ischemia (Fig 1).
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Invasive Hemodynamics and Vascular Wall Elastic Properties: Effects
of Drugs
Hemodynamic, geometric, viscoelastic, and rheological
baseline
values of the abdominal aorta elicited no significant changes in
minipigs on the control diet regardless of whether they were treated
with the captopril-hydrochlorothiazide combination at the dosage
indicated in the present study.
Minipigs on the methionine-rich diet
presented increased systolic
(+27.6%), diastolic (+19.2%), mean (+21.2%), and pulse
(+52%)
aortic blood pressures and heart rate (+30%) compared with control
animals (Table 4). In M+Cp animals, diastolic blood
pressure returned close to control levels, whereas systolic and mean
blood pressures were higher compared with the C group, although
systolic blood pressure was lowered compared with the M group. These
invasive results, therefore, are in agreement with noninvasive
plethysmographic observations. Animals in the M group had significantly
decreased mean blood flow (-19.6%) and increased pulse flow
(+33.4%)
compared with the C group (Table 4). The hysteresis loop
representative of the pressure-flow relation in the M group
(Fig 2) shows that increased blood pressures in the
hyperpulsatile abdominal aortas were associated with decreased
diastolic blood flow (-20.5%) and, to a lesser extent, increased
systolic blood flow (+8.1%), which, although not statistically
different in absolute values from control pressures, combined to
account for significant mean blood flow decrease and pulse flow
increase in animals in the M group compared with the C group. There
were no statistical differences in flow values between the M+Cp group
and either the C, M, or C+Cp groups. These changes in hemodynamics
resulted in part from changes in hind-limb peripheral resistances that,
compared with animals in the C or C+Cp groups, largely increased in the
M group and returned to normal in the M+Cp group, thus accounting for
the normal diastolic blood pressure in the M+Cp group. As a result,
from the parallel and concomitant changes with pulse flow and pressure,
there was no significant difference in aortic input impedance between
animal groups (Table 4).
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The geometry of minipig
abdominal aorta in the M group was
significantly altered from that of control animals, showing
concomitantly increased systolic, diastolic, and mean external
diameters (+17%) and dramatic doubling of pulse diameter (+104%)
(Table 4
). The mean wall thickness/radius ratio was not
significantly
changed in the M or M+Cp groups compared with the C or C+Cp groups
because of parallel increases in wall thickness and radius. In
contrast, the animals in the M and M+Cp groups had elevated ratios of
diastolic-systolic wall thickness difference to mean radius (80.4%
and 23.3%, respectively), which indicated an attenuation of the
vascular wall thickness flattening during the systolic rises in blood
flow and pressure in M and M+Cp minipigs compared with C animals (Table
4). The aortic enlargement and marked lengthening of the
hysteresis
loop representative of the diameter-pressure relation in the
M group (Fig 3) depict the mega-artery syndrome found in
the hyperhomocysteinemic animals. The abdominal aorta geometry was
significantly less altered in minipigs of the M+Cp than of the M group,
as reflected by lower mean, external, and pulse diameters (Table
4) and
a moderate lengthening of the diameter-pressure hysteresis loop (Fig
3).
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The hysteresis loops relating the aortic blood
pressure-external diameter and blood pressure-flow relations
in the C, M, and M+Cp groups depict the deleterious
hyperhomocysteinemia-induced changes in the elastic behavior of the
aorta and the preventive effects of the treatment by the
captopril-hydrochlorothiazide association as well (Figs 2 and
3).
Compared with the C group, the rheological and viscoelastic parameters
were strikingly altered in the abdominal aortas of M group
minipigs by an increase in volumetric compliance (+42%) and
midwall stress (+29%) and by a reduction in aortic stiffness (-25%)
and Young's elastic modulus (-22%) (Table 4). The
increased aortic
compliance, which reflects the behavior of the aorta as a hollow
structure submitted to the blood pressure deforming stress, accounts
for the hyperpulsatility of the aorta in the M group. The lowered
aortic stiffness and Young's elastic modulus, which reflect the
magnification of the resulting strain in the wall induced by the blood
pressure deforming stress, indicate that the intrinsic elasticity of
the aortic wall has diminished in the M group and therefore accounted
for the increased volumetric compliance. Compared with animals in the M
group, the M+Cp animals presented much less altered aortic
volumetric compliance and midwall aortic stress, which returned to
control levels, whereas elastic modulus and aortic stiffness remained
significantly different from the C group (Table 4). The aortic
compliance recovery in the M+Cp group therefore reflects the
maintenance by the drug association of a normal pulsatility in the
abdominal aorta in this group. However, the aortic stiffness and
Young's elastic modulus of the aortic wall remained altered, thereby
revealing the remaining presence of a resulting elevated strain in the
wall in response to blood pressure stress (Table 4). In the
M+Cp group
(Fig 3), the retention of an inflection area in the ascending
limb of
the hysteresis loop representative of the diameter-pressure
relation resulted from the preservation of the initial inertia in
diameter change as blood pressure increased. It thereby illustrated the
preventive effects of the drugs on the elastic component of the
vascular wall rheology in these animals.
Histopathology of Vessels: Effects of Drugs
At histological
examination, the adult control animals were found
to be not entirely free of lesions, since their LIVCA, and to a lesser
extent abdominal aorta, exhibited few vascular streaks, essentially
fibrosclerotic in nature (data not shown), typically resembling those
of aging vessels.34 38
In the M group, the
abdominal aorta (Fig 4A and 4B),
carotid bifurcation (Fig 5A and 5B), LIVCA (Fig
6A and 6B), and peripheral muscular arteriolae
(Fig 7A) showed pronounced fibroelastic disorders associated
with marked smooth muscle cell alterations. The lesions in capacitance
arteries were always present, spreading along the longitudinal axis
of the arteries with specific patterns of development according to the
arterial site. Resistance arteries were either consistently affected
for most of them or unscathed by disease for the remaining few. Aortic
and carotid lesions showed intravascular wall development of disease
and were barely protruding within the luminal space, whereas coronary
lesions simultaneously bulged into the vascular lumen and progressed in
depth in the arterial wall while preserving the deeper layers of the
coronary media. At the lesion site, hypertrophic endothelial cells
covered a thickened subendothelial space by the cumulative presence of
edematous changes and fibrous deposits. In the aorta and carotids,
internal elastic laminae and deeper elastic laminae were prominently
split and disrupted, and amounts of fragmented, disorganized elastic
fibers and collagen were observed. Hypertrophy and reorientation of
medial smooth muscle cells were constant pathological features of
lesions. Hyperplasia, commonly associated with elastic lamina
disruption, was detectable in focalized loci. The disruption of elastic
laminae led to the settlement of communicating pathways between
muscular laminae, which enabled cell spreading processes to occur (Figs
4A, 5A, and 6A). In the coronary
arteries, hypertrophy and migration of
the smooth muscle cells were associated with dramatic hyperplasia and
caused the lesion to bulge into the lumen because of modulated smooth
muscle cells invading processes of the subendothelial space through the
fragmented internal elastic laminae (Fig 6B). The invasion of
subendothelial space by migratory smooth muscle cells was prominent in
coronary arteries compared with aortas and carotid arteries.
Insignificant lipid deposition was observed within all the lesions. The
peripheral resistive arteries of the minipig legs in the M group
presented with marked lumen reduction and medial cell hypertrophy
and, to a lesser extent, hyperplasia (Fig 7A).
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Arterial
lesions were still observed in the vessels of
captopril-hydrochlorothiazide–treated minipigs (group M+Cp), but
with
substantial, specific differences compared with untreated animals. In
the abdominal aorta (Fig 4C), carotids (Fig 5C),
and LIVCA (Fig 6C) of
M+Cp animals, alterations of internal elastic laminae and underlying
elastic laminae were moderate, and the normal stacking up of muscular
laminae was preserved. The settlement of cell communicating pathways
commonly found between muscular laminae in the M group was rarely noted
in the M+Cp group. In contrast, smooth muscle cell hypertrophy was
still present in the M+Cp group, being moderately lowered compared
with the M group, whereas foci of cell hyperplasia were no longer
detectable in the media in the M+Cp group of animals. Hypertrophic
smooth muscle cells were present in the vascular wall of resistive
arteries (Fig 7D). However, arteriole vascular walls were not
observed
to be prominently thickened as in animals in the M group, and the
lumen/wall thickness ratio of these arterioles was persistently found
to be <0.5 in the M+Cp group, whereas it was regularly >0.5 in the
peripheral arteries of the animals in the M group.
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Discussion |
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TopAbstractIntroductionMethods Results Discussion References |
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Hyperhomocysteinemia in Minipigs
We report here that minipigs fed a caseinate-enriched diet over the long term presented within at least 24 hours with lasting substantial hypermethioninemia, hyperhomocysteinemia, and a tendency to have increased plasma
-aminobutyrate levels without excess or
deficiency in other plasma amino acids as well as disorders in serum
parameters of electrolytic, lipid, and glucose metabolisms. The minipig
was the species selected because results from sulfur amino acid
metabolism experiments in the latter, which involve rates of metabolite
flux, more closely match the results obtained in humans than do those
from rabbits and rats (see Reference 39 for review).
The elevated
plasma homocysteine and -aminobutyrate, which are
characteristics of the transsulfuration pathway, provide evidence that
hyperhomocysteinemia results from the overloading of the
transsulfuration pathway, although both transaminative and
transsulfuration pathways of L-methionine metabolism are
approximately equivalent in humans and pigs.39 Previous
results in minipigs on a semisynthetic diet based on either casein or
soy protein isolate have shown that the venous postprandial amino acid
profile reflects differences in the amino acid composition of the
protein consumed. Specifically, a unique group of eight amino acids
(methionine, arginine, tyrosine, valine, tryptophan, leucine, lysine,
and cysteine) evidenced significant protein-dependent quantitative
differences during the postprandial phase, whereas all protein amino
acids developed venous plasma concentration changes in qualitative
accordance with their content in the dietary protein
consumed.19 In animals on a casein-based diet compared
with a soy protein–based diet, methionine postprandial plasma
concentrations elicit a threefold increase, substantially higher than
that of all the other plasma amino acids.19 It is
therefore likely that hyperhomocysteinemia originated from the elevated
L-methionine content of caseins, 2.5-fold that of soy
protein.19 30 In the present study, purified calcium
caseinates from bovine milk were used instead of caseins because
caseins are insoluble and poorly digestible and contain several
non–methionine-rich contaminants such as phosphoproteins, lipids, and
carbohydrates, which may bias the study by unidentified specific
additional and/or adverse effects.39
The concentrations in plasma homocysteine recorded here, ranging from 6.5 to 22.2 µmol/L, are of the same kind as levels found in patients with mild hyperhomocysteinemia and are thought to contribute to their premature vascular disease.1 4 5 In addition, hyperhomocysteinemia developed in minipigs without significant changes in serum lipid profile reflecting hypercholesterolemia. This feature is acknowledged in the minipig, whereas hypercholesterolemia develops in rabbits on a hyperproteinemic diet in a species-dependent manner.19 Our findings reproduce the situation encountered in young homocysteic patients whose serum lipid profiles usually do not reveal hypercholesterolemia5 and in whom arteriosclerotic lesions contain little or no evidence of accumulated lipid in foam cells or extracellularly.40 The present results in experimental hyperhomocysteinemic minipigs, therefore, are in accordance with the conclusions that homocysteinemia could represent a nonlipid model for the initiation of arteriosclerosis.40
After 4 months of the present protocol, two untreated hyperhomocysteinemic pigs underwent major cardiovascular events that finally resulted in thromboembolic syndromes. Although the thrombogenic mechanisms of hyperhomocysteinemia are not well understood, marked platelet accumulation at sites of vascular injury, platelet-rich occlusive thrombi, and thromboembolism are distinctive pathological features in both human and experimental hyperhomocysteinemia.10 41 The endothelial cytotoxicity of homocysteine,7 11 41 42 by exposing subendothelial connective tissue, represents a possible mechanism for platelet activation and homocysteine-induced thromboembolism.10 41 However, the absence of lipid peroxide excess in homocysteic patients, reflecting a low generation of oxygen peroxides generated through the chemical reactivity of the thiol group, opposes the concept that homocysteine-derived peroxides may cause thrombotic disorders.40 Conversely, it has been shown that homocysteine inhibits transport of thrombomodulin through the endothelial cell secretory pathway, resulting in decreased cell surface expression.43 Selective inhibition of the intracellular transport of von Willebrand factor and thrombomodulin, therefore, could predispose to thrombosis by altering the balance between procoagulant and anticoagulant proteins. Thromboembolic disorders also may be magnified by the loss of the capacity of the endothelium (in hyperhomocysteinemic animals) to abolish the toxicity of sulfur-containing amino acids through the formation of S-nitrosothiol from endogenously derived nitric oxide.42 The occurrence of thromboembolic disorders in the minipigs of the present study argues further for the pathological relevance of the experimental hyperhomocysteinemia reported here.
Consequences of Hyperhomocysteinemia for Hemodynamics and Vascular
Rheology and Reactivity
Hyperhomocysteinemic minipigs presented a
mega-artery syndrome
and hyperpulsatile arteries characterized by (1)
systolic-diastolic hypertension originating in part from
elevated peripheral resistances, (2) lowered mean blood flow and raised
pulse blood flow due to the failure of moderately increased systolic
flow to compensate for markedly decreased diastolic flow, and (3)
parallel enlargement in wall thickness and aortic midwall radius, with
a dramatic doubling of pulse diameter. The dramatic strengthening in
pulse diameter resulted from a marked increase in vascular volumetric
compliance. The latter originated from the lowering in the intrinsic
elasticity of the aortic wall, as disclosed by the curtailment in
aortic stiffness and decreased "elastic response," ie, the
-Ep
couple,24 37 due to Ep shift toward lower values. In
contrast, the "stress response," represented by the
-R relation,24 37 was highly amplified in M
animals,
clearly indicating an increase in vascular smooth muscle tension. In
hyperhomocysteinemic animals, the enlargement along the long axis and
the trapezoidal-to-ellipsoid changes in the shape of the
pressure-diameter and pressure-flow hysteresis loops illustrate that
the amplitude of the viscoelastic response was dramatically increased
in these minipigs. The loss of inertia within the diameter and flow
responses to early changes of systolic blood pressure turn the vascular
wall into a soft tube in which the intrinsic elastic component is
markedly altered and the viscous component becomes
dominant.37 44 45 In the physiological
range of blood
pressure and parietal strain, the elastic component accounts
prominently for the wall response to the distending blood pressure and
permits much of the necessary elastic recoil of the aorta against the
pulse pressure.45 46 In the present study, we
demonstrate that the quick elastic component of the vascular response
to tension (which corresponds to blood pressure in the capacitance
vessel) is markedly altered by hyperhomocysteinemia, leading to the
prevalence of the viscous component, which accounts for the delayed
relaxation44 and the imbalanced response of capacitance
arteries to blood pressure.
After transitory caudal circulation arrest, the untreated, hyperhomocysteinemic minipigs presented an extended reactive hyperemia both in an expansion of the increased blood flow and a lengthening of the hyperemia period, which indicate that vascular reactivity was amplified in the animals. Recent studies of reactive hyperemia in the circulation after selective damage to the endothelium or in the presence of nitric oxide synthesis inhibitors suggest that endothelium-derived relaxing factors (EDRFs) may partially contribute to the response.47 Postocclusion reactive hyperemia may also be mediated by a local reflex involving activation of sensory C nervous fibers leading to the release of neuropeptides (calcitonin gene–related peptide and substance P) associated with local vasodilation and increased blood flow.35 48 49 50 In homocysteinemic minipigs, it is therefore conceivable that altered endothelium and/or excited sensory nerves stimulated to release excessive amounts of peptidic vasodilators mediate part of the subsequent sustained reactive hyperemia.
Consequences of Hyperhomocysteinemia for Vascular Wall
Histology
In untreated homocysteinemic minipigs, hypertrophic
endothelial
cells covered an edematous and fibrous subendothelial space. Elastic
lamina disruption resulted in the settlement of communicating pathways
between muscular laminae and spreading of reoriented smooth muscle
cells. In LIVCA, these processes caused the lesions to bulge out in the
lumen while preserving the inner layers of the coronary media. In
contrast, lesions in aortas and carotids had an in-depth parietal
development only. The peripheral microvessels also presented a
marked lumen reduction due to medial cell hypertrophy and, to a lesser
extent, hyperplasia. Insignificant lipid deposition was observed within
all the lesions. Homocysteine-induced lesions therefore elicited the
histopathological features of the arterial wall in hyperhomocysteic
human patients6 20 21 as well as the
specific organization
of atherosclerotic lesions also found in similar regions of human
arteries.38 44 51 52 53 54
The link between homocysteine and elastic lamina fragmentation and splitting, smooth muscle cell hypertrophy, hyperplasia, and migration remains largely unknown. With respect to the homocysteine toxicity against vascular cells,10 it would not be surprising to find that priming of vascular cells by homocysteine results in release of cytokines and/or growth factors by the injured cells, as is the case after balloon-induced vascular injury.55 Specifically, smooth muscle cell activation and replication in the media may be driven by basic fibroblast growth factor released by injured medial cells,55 although other known and unknown growth factors may also be involved. It is also likely that the migration response is linked to the production of a platelet-derived growth factor (PDGF) that has more chemotactic than mitogenic properties in the balloon-injury model.56 PDGF is presumably released within the vascular wall, because platelet activation and deposition in the vascular wall are common features with human and experimental hyperhomocysteinemia.10 41 In the vascular wall of hyperhomocysteinemic minipigs, it is evident that cell spreading processes were favored by elastic lamina fragmentation. One of the key factors in understanding the mechanisms governing these processes is presumably the observation that localized elastic fiber splitting occurred in the presence of focalized smooth cell hyperplasia. Dividing secretory smooth muscle cells had the capacity to release protease activities, namely elastases and collagenases.56 57 Activation of latent collagenase and elastase facilitates migration by proteolytic degradation of the extracellular matrix, freeing cells and permitting their movement in response to chemoattractant stimuli.57 The similarities between homocysteine- and balloon injury–induced alterations of the vascular wall will serve as a framework for a working hypothesis to elucidate the mechanisms accounting for the vascular consequences of hyperhomocysteinemia.
Effects of the ACE Inhibitor–Diuretic Combination on
Hyperhomocysteinemia-Induced Changes
Our results demonstrate that the
captopril-hydrochlorothiazide
association removed diastolic hypertension and prevented disappearance
of the vascular elastic structures and intrinsic elastic component,
which resulted in the maintenance of the trapezoidal shape of the
aortic pressure-diameter hysteresis loop. In contrast, the treatment
poorly prevented vascular cell hypertrophy and wall tension increase
and kept the wall viscous component and systolic hypertension elevated
in animals in which peripheral resistances had returned to normal. On
the basis of the ACE inhibitor–diuretic association, the vascular
therapeutic approach has a protective effect made evident by the
absence of death, detectable tissular infarction, and thromboembolic
disorders in treated minipigs. However, the drugs have no metabolic
effects, since they failed to normalize hyperhomocysteinemia. If we
extrapolate our present results to the human situation, the
beneficial effects of captopril and hydrochlorothiazide in a
hyperhomocysteinemic animal model having the metabolic, structural, and
functional characteristics of vessels in homocysteic patients therefore
favor the use of the combined ACE inhibitor–thiazide diuretic
therapy.
In treated hyperhomocysteinemic minipigs, the decreased blood pressure and increased blood flow resulted from the peripheral arteriolar vasodilation due to the inhibition of generation of the vasoconstrictor angiotensin II and degradation of the vasodilator bradykinin with subsequent release of NO/EDRF, as well as to the adrenergically driven lowering of muscle tone by blocking the release of norepinephrine from the perivascular nerve endings.58 59 However, difficulties arise in estimating the respective contributions of hemodynamic and tissue-based effects of antihypertensive drugs to the overall therapeutic effects. The decrease in blood pressure per se modifies the function of the capacitance arteries by a passive decrease in arterial diameter and volume, an increase in arterial compliance, and a proportional decrease in systolic and diastolic pressures.44 The intrinsic effects of blood pressure lowering on the behavior of capacitive arteries therefore are in clear opposition with our findings that treated hyperhomocysteinemic minipigs had increased vascular wall tension, reduced aortic compliance, and diverging changes in diastolic and systolic hypertension. The beneficial effect of ACE inhibitor-thiazide association resulted from specific actions of the drugs on the aortic wall, instead of passive parietal improvements due to an action on the small resistance vessels and subsequent blood pressure lowering. This view is further supported by previously reported studies comparing the effects of ACE inhibitors with other vasodilating agents (eg, hydralazine and verapamil) that were devoid of antiproliferating properties even though they lowered blood pressure.59
Throughout the study, the treated minipigs showed the inhibition of serum ACE activity usually encountered in captopril-treated patients22 23 29 and, by the end of the protocol, showed both an inhibition of tissue ACE activity and a significant blood pressure reduction. It is likely that the values reported here for ACE inhibition underestimate the functional ACE inhibition, since the rapid rate of ACE inhibitor dissociation made the enzyme become disinhibited during the time taken to assay ACE activity biochemically.32 The daily low dose of captopril that blocked serum and tissue ACE activity in the treated minipigs was selected to keep an ACE inhibitor dosage schedule that might be used in the clinical setting. As previously discussed in reports of the therapeutic effects of perindopril in atherosclerotic minipigs,24 it is indeed questionable that the drug association may elicit more pronounced therapeutic effects when used with higher dosage schedules because higher ACE inhibitor dosages may aggravate the progression of atherosclerosis in minipigs.
The widely used combination of an ACE inhibitor with a thiazide diuretic has synergistic antihypertensive effects and prevents the reciprocal drug-induced stimulation of the renin-angiotensin-aldosterone-potassium retention axis.28 29 60 However, the rationale for considering the captopril-hydrochlorothiazide association also rested on emerging data supporting the view that hydrochlorothiazide, alone or in combination with captopril, may have beneficial vascular properties. In stroke-prone spontaneously hypertensive rats (SHR), long-term administration of hydrochlorothiazide induced a marked prevention of interstitial and perivascular fibrosis independently of blood pressure–related processes.61 The association of captopril plus hydrochlorothiazide may lower the intracellular Na/K ratio in medial cells, thus diminishing smooth muscle contractility,62 and, in pithed SHR rats, may decrease the aortic pulse wave velocity and thereby increase aortic distensibility independently of blood pressure.28 However, the mechanisms accounting for the amplifications of the properties of ACE inhibitors by hydrochlorothiazide that improve the functioning of capacitance arteries in homocysteinemic animals, as well as in hypertensive rats22 and humans23 and in atherosclerotic minipigs,24 25 26 27 remain to be elucidated.28
Although the cellular mechanisms accounting for the beneficial effects of ACE inhibitors against atherogenesis and neointima formation after balloon injury are not clearly understood, the drug is thought to act on these processes by blocking one or several uncontrolled mechanisms in the autocrine and paracrine functions the renin-angiotensin system exerts on the arterial wall.63 Specifically, the findings that the effects of ACE inhibitors are reduced by >50% by the kinin B2-receptor antagonist Hoe 140 and furthermore are blocked by the NO synthesis inhibitor nitro-L-arginine methyl ester59 64 strongly suggest that the protective effect of ACE inhibitor is due to both blockade of angiotensin II formation and kinin degradation. Moreover, NO/EDRF may play a major role in the inhibitory effect of ACE inhibitors.59 The action of ACE inhibitors against hyperhomocysteinemia-induced smooth muscle cell hyperplasia and migration therefore may involve removal of a progrowth influence (angiotensin II) and increase of an antigrowth influence (kinins/NO).32 42 In contrast, very little is known about the destruction of medial elastin underlying the hyperhomocysteinemia-induced structural and functional alterations of vascular walls.7 41 With regard to homocysteine toxicity against vascular cells and increased wall tension in homocysteinemic minipigs, it is conceivable that the release of activable elastinolytic enzymes from vascular cells was induced by stretch and strain or resulted from cell death.65 In addition, the continuous activation of endogenous vascular elastases during disease progression may also result in enhanced susceptibility to elastases.65 The mechanisms accounting for the preventive effects of ACE inhibition against elastic fiber degradation are unknown but might be related to inhibition of plasma and/or tissue angiotensin II effects on the structural component of the vascular wall.66 As previously emphasized from inhibition by ACE inhibitor of the elastic feature degradation in atherosclerotic minipigs,27 one cannot rule out the possibility that captopril, a protease inhibitor, inhibits one or several elastinolytic enzymes. Since the elastic fibers subjected to degradation are not resynthesized in a normal way, thus providing fibers inappropriate for normal function,67 it is likely that the preventive effects of ACE inhibition on elastic fiber alterations will have major functional consequences favoring arterial blood circulation.
Conclusions and Implications of the Study
To sum up, we have
shown that it is feasible to induce an
efficient diet-induced hyperhomocysteinemia in minipigs that reproduces
the metabolic, histopathological, and thromboembolic life-threatening
situation encountered in homocysteic patients. This is the first report
of an experimental hyperhomocysteinemia having a pathological
relevance. Attempting to incorporate the present results into our
understanding of the causes of cardiovascular disease, it might be
relevant to consider that calcium caseinates (as a unique source for
dietary protein in the presence of a balanced vitamin supply) magnify
the usual casein-induced postprandial hypermethioninemia and lead to
hyperhomocysteinemia by overloading the methionine transsulfuration
pathway. Further studies are required to understand the mechanism(s)
for this phenomenon to occur in vivo. Finally, the present results
confirm the preventive therapeutic effects shared by ACE inhibitors,
associated with hydrochlorothiazide, against the activated
elastinolytic processes in the arterial wall. Since mild
hyperhomocysteinemia, as a metabolic disorder, is prone to be
normalized by vitamin therapy, our findings that the ACE
inhibitor-diuretic combination has therapeutic effects on the vascular
wall therefore lead to the possibility that either the therapeutics may
have additional effects or the vascular therapeutic may be a useful
alternative when vitamin therapy fails to normalize
hyperhomocysteinemia.
Received June 14, 1994; revision received August 25, 1994; accepted September 23, 1994.
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