© 1999 American Heart Association, Inc.
Vascular Biology |
Influence of Elastin Gene Polymorphism on the Elastin Content of the Aorta
A Study in 2 Strains of Rat
From INSERM U 460, UFR de Médecine Xavier Bichat, Paris, France.
Correspondence and reprint requests to Dr M.P. Jacob, INSERM U 460, UFR de Médecine Xavier Bichat, 16 rue Henri Huchard, 75870 Paris cedex 18, France. E-mail jacob{at}bichat.inserm.fr
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Abstract—The elastin content in the thoracic aorta of male Brown-Norway (BN) rats is 31.4±1.2% (dry weight), whereas that of male LOU rats is 37.2±1.0%. A similar difference in the elastin content of the thoracic aorta is also observed in female animals. Furthermore, in the thoracic aorta of young, growing rats as well as in cultured aortic smooth muscle cells, the steady-state level of elastin mRNA is significantly lower in the BN than in the LOU strain. These results suggested that 1 or more genes control the elastin mRNA level and the elastin content in the aortas of BN and LOU rats. A possible relationship between a polymorphism in the elastin gene and the elastin content of the aorta was tested. For this purpose, the aortic elastin content was measured in F1 and F2 generations bred from LOU and BN rats and was compared with that of the F0 (parental) generation. A polymorphic marker located in intron 25 of the elastin gene has been used to genotype the F2 rats. The degree of genetic determination of aortic elastin content was estimated to be 73% in the F2 cohort, but the elastin locus accounts for only 3.9% of the total variance in aortic elastin content. Other genes are thus responsible for the major part of the observed interstrain difference by regulating the transcription of the gene, the stability of elastin mRNA, and/or posttranslational events.
Key Words: elastin • genetics • aorta • rats
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Introduction |
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The deposition of functional elastic fibers in arteries during development and growth is a complex process with the possibility of regulation at many levels. Synthesis of tropoelastin and glycoprotein molecules is followed by the relevant organization of these molecules in the extracellular space and the subsequent formation of cross-links between tropoelastin molecules.1 2
Tropoelastin synthesis is the result of the expression of 1 gene and appears to be subjected to complex regulatory processes. Developmental regulation of elastin gene expression results in careful control of the chronology of elastin synthesis during embryonic and postnatal growth, which is also tissue and site specific.3 4 5 6 The mechanisms of this regulation are not yet fully understood. Many factors have been shown to regulate elastin synthesis, such as mechanical factors, cytokines, growth factors, hormones, pharmacological agents, and the composition of the extracellular matrix.3 4 5 6 In essentially all of the in vitro and in vivo studies, changes in elastin synthesis have been shown to be correlated with changes in steady-state elastin mRNA (mRNAE) levels.
Study of the possible genetic control of elastin synthesis had not been previously attempted because no suitable animal model was available. Recent studies on knockout mice have shown that complete deletion of the elastin gene results in obstructive arterial disease, which leads to death 4 days after birth.7 In addition, the elastin gene is implicated in human arterial pathology, since patients with supravalvular aortic stenosis and Williams syndrome have been shown to present mutations or deletions involving the elastin gene on chromosome 7. Histological studies have shown that these patients present disruption of arterial elastic fibers and intimal proliferation in their large elastic arteries, suggesting that a quantitative and/or qualitative modification in elastin during vascular development is of pathogenetic importance.8 9
We have previously characterized a strain of rat, the Brown Norway (BN), which is highly susceptible to spontaneous rupture of the internal elastic lamina (IEL),10 and have shown that this susceptibility to IEL rupture is accompanied by an aortic elastin content significantly lower (by 8% dry weight) than that of the Long Evans (LE) rat, which is resistant to IEL rupture.11 This interstrain difference in aortic elastin content was observed both in the thoracic aorta, which is devoid of ruptures, and in the abdominal aorta, where numerous ruptures occur in the BN rat, suggesting that it represents a general property of arteries in this strain. Quantification of mRNAE in aortas of young, growing BN and LE rats by Northern blotting showed that elastin transcript levels were 2-fold lower in the BN than in the LE strain, in both thoracic and abdominal segments.11 This result suggested that the elastin deficit in the BN rat was, at least in part, the result of decreased mRNAE levels in the aorta during the period of rapid growth. Although in this study we were unable to conclude the existence of a direct relationship between elastin deficit and the susceptibility to IEL rupture, these results nevertheless provided us with an interesting model for the study of the possible influence of genetic factors on elastin synthesis. In view of the role that an elastin deficit or elastin/collagen imbalance may play in predisposing to various vascular pathologies,12 13 14 the question of the genetic control of arterial elastin content is of some importance.
As a first step in the study of possible genetic control of elastin content, we have investigated here whether a polymorphism in the elastin gene is associated with the differences in mRNAE level and elastin content in the aortic wall between 2 inbred strains of rat. Because the inbred LE strain is no longer available, we used the LOU strain and compared it with the BN strain. The LOU strain presents the same characteristics as the LE strain in terms of susceptibility to IEL rupture,15 and preliminary experiments showed it to have a significantly higher aortic elastin content than the BN strain. We have thus quantified aortic elastin content in F1 and F2 generations bred from LOU and BN rats and compared it with that of the F0 (parental) generations. A polymorphic microsatellite located in intron 25 of the elastin gene has been used to genotype the F2 rats16 and to test whether the BN allele is linked to a lower elastin content than the LOU allele. The mRNAE level was also measured in thoracic aortas and cultured smooth muscle cells of F0 rats as well as in the thoracic aortas of F1 and F2 animals.
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Animals
Inbred BN rats were supplied by Iffa Credo (Domaine des Oncins, l'Arbresle, France), and the inbred LOU rats were provided by our own breeding stock. Female LOU rats and male BN rats were mated to produce F1 hybrids. Male and female rats of the F1 generation were mated to produce the F2 cohort.
Animal care complied with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication No. 86-23, revised 1985). The studies were carried out under authorization No. 006235 of the Ministère de l'Agriculture, France.
Blood Pressure Measurements
Systolic arterial blood pressure was
measured on conscious animals under standardized conditions routinely
used in our laboratory by using a tail cuff and pulse transducer (BP
recorder 8006, Apelex) after 20 minutes under a specially designed
heating device at 32°C. Systolic arterial blood
pressure and heart rate were determined on 3 successive days in BN and
LOU rats aged 6 weeks.
Quantification of Aortic Elastin
The quantity of elastin present in the thoracic aorta was
measured in mature adult rats (18 weeks old; 6 male and 6 female BN and
LOU rats, 6 male and 6 female F1 rats, and 119
male and 42 female F2 rats). After body
weight was recorded, the rats were killed and the hearts and
descending aortas (from the aortic arch to the diaphragm) were
dissected out. Heart weights were recorded, and thoracic aortas
were cleaned of blood and surrounding adipose tissue in ice-cold
saline, frozen in LN2, and stored at -80°C
until biochemical analysis.
Just before biochemical analysis, aortas were thawed and manipulated in a Petri dish on ice under a dissecting microscope. The aortic arch was discarded, the remaining segment was opened longitudinally, and its length was recorded by using a grid in the eyepiece. Elastin was then quantified by using a method based on that described by Wolinsky,17 as follows. After delipidation in acetone/diethyl ether (1:1, vol/vol) and drying, the dry weight was recorded by using a Sartorius R 160P balance (precision of 0.01 mg). Cell proteins were extracted by gentle agitation in 0.3% SDS for 12 hours. The extracellular proteins other than elastin remaining in the aortic segments were solubilized by three 15-minute extractions in 1 mL of 0.1 mol/L NaOH in a boiling water bath. Elastin was quantified by determining the dry weight of the residue. The purity of this elastin was confirmed by amino acid analysis (data not shown).
Quantification of Other Aortic Constituents: Cell Proteins and
Collagen
Total cell protein was assayed in the SDS extract by the method
of Lowry et al.18 Aortic collagen content was determined
by assaying the hydroxyproline present in the NaOH solution. NaOH
solutions were evaporated to dryness and hydrolyzed in 6N HCl in
vacuum-sealed vials for 24 hours at 110°C. Hydroxyproline was
determined in the hydrolysate by using a colorimetric
assay according to Woessner.19 Collagen was quantified
from hydroxyproline values on the basis of the assumption that collagen
contains 12.77% hydroxyproline by weight.20
Analysis of mRNAE by Northern Blot
By Northern blotting, steady-state levels of thoracic aortic
mRNAE were evaluated in young, growing rats (6
weeks old) and in young adult rats (12 weeks old). Six-week-old and
12-week-old male and 6-week-old female rats of both strains (n=10 in
each group) were used for this purpose. Aortic
mRNAE levels were also measured in 10
F1 and 40 F2 male rats aged
6 weeks.
At the time the animals were killed, the thoracic aorta between the
heart and the diaphragm (including the ascending aorta and aortic arch)
was dissected out, rapidly cleaned of any adipose tissue in ice-cold
saline, and immediately frozen in LN2. They were
stored at -80°C until used. Total RNA was isolated by the
guanidinium isothiocyanate/phenol extraction procedure.21
Samples were homogenized with a Kinematica Polytron in 5
mol/L guanidinium thiocyanate solution. Total RNA (in 19% glyoxal,
50% dimethyl sulfoxide, and 0.01 mol/L sodium phosphate buffer) was
fractionated in a 1.2% agarose gel (18 mA, 4°C, for 16 to 18 hours).
RNAs were then transferred to a nylon membrane (Hybond, Amersham). A
cDNA probe for rat elastin, named REL-124D,22 was used to
detect mRNAE. The elastin probe was labeled by
random oligonucleotide primer extension with
[
-32P]dCTP (NEN-Dupont). The hybridization
solution contained 50% deionized formamide, 1% SDS, 1x Denhardt's
solution, 0.05 mol/L sodium phosphate buffer, 250 µg/mL salmon sperm
DNA, and 5x SSC. The probe was heat-denatured, and 200 000 counts per
minute were added to 100 µL of hybridization solution. Hybridization
was performed overnight at 42°C with 100 µL of solution for each
square centimeter of membrane. Three washes followed: 2 in 2x SSC,
0.1% SDS at room temperature for 15 minutes and 1 in 0.1% SSC, 0.1%
SDS at 60°C for 20 minutes. The membrane was then exposed to
autoradiographic film with an amplifying screen at -80°C
for several hours.
The membranes were then reprobed with a human 28S RNA
oligonucleotide23 to normalize
mRNAE. The primer was end-labeled with
[
-32P]ATP (NEN-Dupont) by using T4
polynucleotide kinase (New England Biolabs). The
hybridization solution contained 4x SSPE, 0.1% sodium pyrophosphate,
and 0.2% SDS. Approximately 106 cpm were added
per milliliter of hybridization solution. After overnight hybridization
at 42°C, the membrane was washed (30 minutes at room temperature in
2x SSPE, 0.1% sodium pyrophosphate, and 0.1% SDS) and reexposed to
film for 2 hours at -80°C.
Quantitative estimates of band intensities in each lane (elastin and 28S hybridization) were obtained after scanning the autoradiograms with a scanner (Agfa Studio Scan II Si) with the use of Adobe PhotoShop software and were quantified by using NIH Image 1.60 software.
Cell Culture
Because local hemodynamic and environmental
parameters may differ between BN and LOU rats and thus
influence elastin synthesis in vivo, the mRNAE
level was also measured in cultured SMCs. SMCs were isolated from
thoracic aortas of BN and LOU rats aged 6 weeks, as follows. The aortas
were quickly excised and rinsed in Hanks' balanced salt solution
(Sigma) containing heparin (25 U/mL, Léo). The adjacent tissues
were removed, the collateral vessels were cut, and the aorta was opened
longitudinally. The intima-media was removed by gentle peeling, cut
into small pieces, and incubated in 5 mL of collagenase
(0.2% [624 U/mg, Eurobio] in DMEM) and elastase (0.06% in DMEM,
Eurobio). After 60 minutes at 37°C under agitation, the SMC
suspension was flushed, filtered through a sterile porous nylon
membrane, washed with 20 mL of warm Hanks' balanced salt solution, and
then centrifuged. The cell pellet was resuspended in culture
medium (DMEM) with 10% FCS (Dutscher), 1x
antibiotic-antimycotic solution (Sigma), and 0.02 mol/L HEPES
(Boehringer Mannheim) and plated in a
25-cm2 plastic flask coated with collagen (1 mL
of a 0.05% solution per 25 cm2). The purity of
the cell culture was tested by immunodetection of SM -actin with a
specific antibody.
At passage 2, 2 days after reaching confluence, cells were incubated in 1% FCS medium for 48 hours and then harvested in 5 mol/L guanidinium-thiocyanate solution for total RNA extraction. The level of mRNAE was then measured by using the Northern blot technique as described above.
Genotyping of F2 Rats
The genotype of each F2 rat was
determined by using the polymerase chain reaction (PCR) technique. For
each animal, genomic DNA was prepared by phenol extraction from a 1-cm
fragment of the tip of the tail. The PCR primers used to amplify a
fragment of the elastin gene, which included the intron 25/exon 26 and
exon 26/intron 26 boundaries, were determined with OLIGO 4
software. Primers were end-labeled with
[-32P]ATP (NEN-Dupont) with the use of T4
polynucleotide kinase (New England Biolabs). One hundred
nanograms of genomic DNA was amplified in a 25-µL PCR, with final
concentrations of each reagent being 75x10-6
mol/L dNTPs (Pharmacia), 0.4 x10-6 mol/L of
each of the 2 primers, 0.6 U of Taq DNA polymerase (Life
Technologies), and 0.001 mol/L MgCl2. Reactions
were performed in a Perkin-Elmer apparatus by using the
following protocol: initial denaturation at 94°C for 4 minutes;
followed by 30 cycles of 94°C for 30 seconds, 62°C for 30 seconds,
and 72°C for 30 seconds. This was followed by a final elongation step
(10 minutes at 72°C). PCR products were submitted to
electrophoresis on 4% polyacrylamide denaturing gel for 4
hours at 65 W. Gels were dried and then exposed to autoradiographic
film overnight at -80°C. Genotypes for
F2 animals were determined by observation of the
length and number of PCR amplification products.
Statistical Analysis
Data are expressed as mean±SD unless otherwise stated. The
Mann-Whitney U test, a 1-factor ANOVA followed by the Fisher
test, and a 2-factor ANOVA were used for statistical analysis
of results as stated in the legends to the figures. The degree of
genetic determination of relative elastin content in the aorta was
estimated as
(Vt-Ve)/Vt,
where Vt is the total variance of this
parameter in the F2 cohort and
Ve is the pooled estimate of the variance
of this parameter in the LOU, BN, and
F1 cohorts (variance due to the effect of
"environmental factors," ie, hemodynamic, hormonal,
soluble, and other undetermined factors, added to experimental errors).
The proportion of elastin content variance in the
F2 rats that could be attributed to the elastin
microsatellite locus in the F2 cohort was deduced
from the calculation by ANOVA of the sum of squares due to the
genotypes compared with the sum of squares of elastin content
in the F2 group.
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Results |
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Results for the Parental Strains: BN and LOU Rats
Aortic Elastin Content
Figure 1
shows elastin content in
the thoracic aortas of BN and LOU male and female rats aged 18 weeks.
Whether expressed as a percentage of the dry weight of the aorta
(Figure 1A) or as milligrams per centimeter of aorta (Figure 1B), BN aortas had significantly less elastin than did LOU
aortas. There were no overlapping values when results were expressed as
percentages of dry weights. The male-female difference observed when
results were expressed as weight per aorta length is explained by the
considerable difference in body size and thus, in aortic caliber
between sexes (see the Table). These
results show clearly that there is an interstrain difference in aortic
elastin content.
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Quantification of mRNAE
Several factors may be of relevance when considering
mRNAE level in the period of rapid growth of the
rat, such as body weight, blood pressure, and heart rate. At 6 weeks of
age, the body weight of BN male rats was significantly greater than
that of the LOU male rats (140.1±7.7 and 77.1±7.1 g, respectively,
P
0.001). Systolic blood pressure was also
significantly greater in BN than in LOU rats (127±6 and 115±5
mm Hg, respectively, P0.01), but heart rate was not
different between the 2 strains (383±35 and 414±21 bpm, respectively,
NS).
Northern blot analysis for mRNAE
(Figure 2) showed that at 6 weeks, ie,
during the period of rapid growth of the animal, elastin transcript
levels (relative to 28S rRNA) in aortas of the BN strain were half
those found in the LOU aortas, in both male and female rats. At 12
weeks, mRNAE level was quantified in male rats
only. The level of mRNAE was greatly diminished
compared with the 6-week-old rats, and the difference between BN and
LOU rats was no longer observed.
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Quantification of mRNAE in SMCs Isolated From BN and
LOU Thoracic Aortas
Northern blot analysis showed a
significantly lower mRNAE level (relative to 28S
rRNA) in SMCs isolated from BN thoracic aortas than in SMCs isolated
from LOU thoracic aortas (Figure
3). Thus, both in vivo and in vitro, BN
aortic SMCs contained lower levels of mRNAE than
those of LOU rats. In view of these results, together with the fact
that adult BN rats presented a lower aortic elastin content
than did adult LOU rats, we went on to test whether different
alleles of the elastin gene could be implicated in the difference
in aortic elastin content.
Results for the F1 and F2
Descendants
Genotyping of the F2 Descendants
Genetic markers enable one to identify the alleles exerting a
genetic effect on a quantitative trait. In an attempt to define the
role of the elastin gene polymorphism in the control of aortic
elastin content, we used a previously described polymorphism at the
elastin gene locus.16 In this study, sequences for the PCR
primers were given. With the use of these primers, the PCR-amplified
product with BN DNA is a 216-bp fragment, but there is no amplified
product with DNA extracted from LOU rats (Figure 4A). Thus, it would be impossible to
distinguish F2 homozygous BN rats from
heterozygous ones by using these primers. Therefore, with the use of
the same polymorphic marker, another primer pair was chosen. The
sequence for the sense primer is the following:
5'-GCA-AAA-CAC-AAA-GGC-ACA-TAA-3'; the sequence for the antisense
primer is the following: 5'-AGT-AAG-GCT-CGG-CAG-TCA-CAT-3'. With DNA
extracted from both BN and LOU rats, the PCR-amplified product with
the use of these primers is a 
560-bp fragment of rat elastin gene,
which includes intron 25/exon 26 and exon 26/intron 26 boundaries.
Unfortunately, it was impossible to separate the 2 fragments obtained
from the DNA of heterozygous rats on a 2% agarose gel (Figure 4B). Therefore, the amplified fragments labeled during the PCR
using an antisense radioactive primer were separated on a 4%
polyacrylamide denaturing sequencing gel. After 4 hours of
migration, it was possible to separate the PCR products (Figure 4C).
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This polymorphic marker was used to genotype all rats of the F2 cohort, comprising 119 males and 42 females. The frequency of genotypes was compatible with a Mendelian mode of inheritance (ie, in the entire F2 population, 23.6% of rats were homozygous for the BN alleles (or BB), 55.3% were heterozygous (or BL), and 21.1% were homozygous for the LOU alleles (or LL).
Aortic Elastin Content in the F1 and
F2 Descendants
Arterial elastin content was measured in
F1 and F2 cohorts and
compared with the results previously obtained for the
F0 cohort. When expressed as a percentage dry
weight (Figure 5) or as weight per unit
length of aorta (Figure 6), mean values
of arterial elastin content in F1
progeny were between those of the 2 parental strains in both male and
female rats.
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In the F2 descendants, arterial
elastin content was compared among the 3 groups defined by the elastin
gene polymorphism. When expressed as a percentage of dry weight,
the mean elastin contents of all male and female
F2 groups were between those of the 2 parental
strains. Homozygous LL rats had a higher elastin content than did
homozygous BB rats in both male and female groups (male LL rats
35.1±2.4%, male BB rats 34.0±2.2%, female LL rats 35.5±1.5%, and
female BB rats 34.3±2.1%; Figure 5). However, the differences
between the BB and LL groups (1.09% for male rats, 1.26% for female
rats) were distinctly lower than the differences between the parental
BN and LOU strains (5.82% for male rats, 6.11% for female rats).
According to the 2-factor ANOVA, the elastin genotype conferred
a significant difference for this parameter among the 3
groups (P=0.043), but the sex of the rats did not. The
elastin contents in the male and female BL groups were closer to those
of the LL groups than to those of the BB groups. The degree of genetic
determination in the F2 cohort was estimated at
73.3% for aortic elastin content. The elastin gene locus accounted for
3.9% of the total variance in elastin content of the
F2 group. When expressed as dry weight per aortic
length, there were no significant differences among the 3 groups of
F2 rats (BB, BL, and LL) in either male or female
groups (Figure 6).
Therefore, there appears to be a low but significant relationship between elastin genotype and the relative content of elastin in the arterial wall.
Quantification of mRNAE in the Aortas of F1
and F2 Descendants
The mRNAE levels (Figure 7) were measured in the aortas of
6-week-old F1 and F2 male
rats. When the mRNAE level (relative to 28S rRNA)
of F1 rats was compared with the
mRNAE levels of parental BN and LOU rats, it was
close to the level of BN rats but significantly lower than the level of
LOU rats. There were no significant differences in
mRNAE levels among the 3 groups of
F2 animals defined by genotyping. Thus, there
appears to be no relationship between elastin genotype and the
quantities of elastin gene transcripts present in the aortas of
6-week-old rats.
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Other Aortic Constituents in Parental Strains and F1
and F2 Descendants: Cell Proteins and Collagen
The Table shows some general data (body weight, heart
weight/100 g body weight, and aortic dry weight) and the results of
quantification of aortic cell proteins and collagen for parental
strains and for F1 and F2
descendants (all aged 18 weeks). This table shows that the body weight
of BN rats was greater than that of age-matched LOU rats, whereas
relative heart weight (expressed per 100 g of body weight) was
greater in LOU than in BN rats. The mean values of body weight and
heart weight/100 g of body weight for male F1
rats were between those of the 2 parental strains. In
F2 rats, there were no significant differences in
body weight among BB, LL, and BL groups in either male or female rats.
However, the heart weight/100 g of body weight was significantly lower
in LL than in BB rats, the mean values for BL rats being intermediate.
In contrast, aortic dry weight (expressed as milligrams per centimeter
of aorta) was not different between the 2 parental strains or among BB,
LL, and BL groups. Cell proteins were significantly higher in BN than
in LOU rats. In the F2 descendants, the results
of cell protein measurements did not show any significant differences
among BB, BL, and LL genotypes in either male or female
rats.
The results for aortic collagen content in the parental strains showed differences similar to those for cell proteins. Aortic collagen content was significantly higher in BN than in LOU rats in both sexes, resulting in a marked decrease in the elastin-collagen ratio in the BN strain. For male F1 rats, mean values for aortic collagen and the elastin-collagen ratio were intermediate to those of the 2 parental strains. In the F2 descendants, aortic collagen content was similar in the BB, BL, and LL groups. The elastin-collagen ratio was higher in the LL groups than in the BB groups, and this ratio was even higher in the BL than in the LL group. The influence of elastin gene polymorphism on the elastin-collagen ratio was almost significant (P=0.07).
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Discussion |
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The results of our study suggest that the elastin gene polymorphism plays a minor but nevertheless significant role in the control of relative elastin content in the rat aorta, because elastin quantity expressed as a percentage of dry weight was significantly different in the BB and LL groups as defined by the different elastin gene alleles. This difference was, however, not as great as the difference observed between the parental strains.
In the parental strains, a marked difference was observed in aortic elastin content between BN and LOU adult rats in both males and females. The lower elastin content of the BN rat at 18 weeks of age could be the result of decreased synthesis, increased degradation, or a combination of both. It could also be the result of a defective arrangement of tropoelastin molecules in the extracellular space due to alterations of microfibrillar glycoproteins. Another possibility is that BN elastin is less cross-linked than that of the LOU strain and therefore more sensitive to solubilization by hot NaOH. Indeed, we have previously shown24 that the BN rat presents a lower lysyl oxidase activity and a higher elastase activity in its aorta than does the LE rat, which is similar to the LOU rat so far as aortic elastin content is concerned. However, we subsequently showed that the difference in elastin content between BN and LE rats can be explained, at least in part, by differences in mRNAE level.11 This is why we compared mRNAE levels in the 2 strains of rat used in this study. Our present results indeed show that the differences in the mRNAE content of aortas of young, growing BN and LOU rats can, in part, explain the differences in aortic elastin content observed in the adult.
The mRNAE level is modulated by several factors, among which hemodynamic factors are considered to be of great importance in the aorta. Increased arterial pressure, via increased wall stress, rapidly increases aortic mRNAE levels and conversely, a decreased arterial pressure decreases mRNAE level.25 For this reason, we measured arterial pressure in the 2 strains of rat to see whether a difference in pressure could explain the observed differences in mRNAE level. Paradoxically, as in our previous study,11 at 6 weeks of age it was the BN rat, which presents the lowest mRNAE level, that had the highest blood pressure. Thus, different blood pressures do not appear to explain the different mRNAE levels at this age. Heart rate did not differ between strains and so this parameter cannot be implicated in the differences in mRNAE level.
It should be noted that at 6 weeks of age, BN rats weighed more than LOU rats. This may suggest that at this age, LOU rats are in a more immature stage of development than BN rats and therefore, may be in a more active phase of synthesis of elastin than the BN strain. Furthermore, the difference in body size, if it represents a difference in maturity, could also explain the observed differences in blood pressure, because blood pressure gradually increases with age from birth until reaching adult levels. However, in our previous study with BN and LE rats, we did not observe such a relation among body weight, blood pressure, and mRNAE level. At 6 weeks of age, LE rats were heavier but had lower blood pressures and higher mRNAE levels than did BN rats.11
It thus appears that the difference in mRNAE level between BN and LOU rats cannot easily be explained by differences in hemodynamic factors. Furthermore, when smooth muscle cells from the aortas of these 2 rat strains were cultured, the cells isolated from BN aortas contained lower levels of mRNAE than did those isolated from LOU rats. Thus, under the same culture conditions and free of any hemodynamic factors, aortic smooth muscle cells isolated from LOU rats intrinsically presented the faculty to synthesize more elastin than did cells isolated from BN aortas. This result strongly suggested that a genetic factor, acting at the cellular level, may play a role in the control of elastin content in the rat aorta and prompted us to look at the elastin gene.
In the F1 cohort, obligatorily BL, the mean value of aortic elastin content was intermediate to the values of the 2 parental strains in both sexes and for both modes of expression (percentage of dry weight or milligrams per centimeter of aorta). For the F2 cohort, the relative elastin content (expressed as a percentage of dry weight) was significantly lower in BB than in LL groups in both sexes. However, no significant difference was observed when the elastin content in the aorta was expressed as weight per aorta length. This lack of significant difference can be easily explained: owing to the large variation in body size and thus, aortic dry weight, among individual F2 rats, the absolute amount of elastin varied widely, and any significant difference in aortic elastin content among BB, BL, and LL F2 rats may have been masked. The expression of elastin content relative to dry weight eliminates consideration of these large individual variations in aortic dry weight.
The determination of mRNAE levels in the aortas of 6-week-old BB, BL, and LL rats did not reveal any significant differences among the groups, in contrast to what was observed for the relative quantity of elastin in the aorta in 18-week-old rats. However, it is possible that subtle differences in mRNAE level throughout the period of rapid growth, which are below the threshold of detection by Northern blotting, may induce significant differences in aortic elastin content in the adult rat.
Analysis of the elastin-collagen ratio in the different F2 groups is of interest, as the difference between groups defined by elastin genotype almost reached significance. Collagen is the most abundant component by weight in the aorta after elastin, and the elastin-collagen ratio is considered to be 1 of the major determinants of arterial distensibility.
In conclusion, a significant difference in aortic elastin content between BB and LL was observed when expressed as a percentage of dry weight of the aorta, whereas no difference was detected for mRNAE between these groups. Thus, both for the relative elastin content in the aorta and for elastin transcripts, the differences between BB and LL are much less than the differences between the parental strains, indicating that the elastin gene polymorphism explains only a small part of the difference between the parental strains. Indeed, according to our estimations, although the total genetic determination of elastin variance in the F2 generation was 73.3%, the elastin gene locus only accounts for 3.9% of this total variance. It is thus probable that some other gene or group of genes is responsible for the major part of the interstrain difference by regulating the transcription of the elastin gene, the stability of mRNAE, the cross-linking of tropoelastin molecules, and/or other posttranslational events.
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Acknowledgments |
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This study was supported by INSERM and the Fondation de France. Marie Sauvage was the recipient of a grant from the Ministère de la Recherche. We thank Liliane Louédec for maintaining the colony of LOU rats and for carrying out the LOU-BN cross-breeding required for this study.
Received November 5, 1998; accepted February 23, 1999.
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References |
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