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(Hypertension. 1995;25:1245-1251.)
© 1995 American Heart Association, Inc.
Articles |
Analysis of the Role of Angiotensinogen in Spontaneous Hypertension
From the Department of Medicine, University of Leicester (UK), Leicester Royal Infirmary (D.L., M.A.K., J.H., N.J.S.); Cardiovascular Department, CIBA-Geigy Ltd, Basle, Switzerland (F.C.); and URA Centre National de la Recherche Scientifique (CNRS) 1483, Faculté de Pharmacie and Laboratoire de Physiologie, Université Claude Bernard (Lyon I), Lyon, France (M.V.).
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Abstract |
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Abstract Allelic variants at the human angiotensinogen locus have recently been reported to increase susceptibility to the development of essential hypertension. In this study we analyzed the role played by angiotensinogen in the elevated blood pressure of the spontaneously hypertensive rat (SHR). The SHR angiotensinogen locus (on chromosome 19) cosegregated with a significant (P=.003) and specific increase in pulse pressure in F2 rats derived from a cross of the SHR with the normotensive Wistar-Kyoto rat (WKY), accounting for 20% of the genetic (10% of total) variance in this phenotype. To identify potential mechanisms underlying the effect of the locus, we further examined angiotensinogen structure and expression in the two strains. Sequence analysis of the respective coding regions revealed no differences in the primary structure of angiotensinogen between the strains. Likewise, plasma angiotensinogen level did not differ in adult rats of the two strains. However, gene expression studies showed tissue-specific, age-related differences in angiotensinogen mRNA levels between SHR and WKY, particularly in the aorta. The findings suggest that pulse pressure, which significantly influences cardiovascular risk, has independent genetic determinants. They further suggest that the effect of the angiotensinogen locus on this phenotype in the SHR may be mediated through a tissue-specific abnormality of angiotensinogen gene expression.
Key Words: renin-angiotensin system • hypertension, spontaneous • genetics • rats, inbred SHR
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Introduction |
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Blood pressure (BP) is a multifactorial trait with both genetic and environmental determinants. Hypertension, a major risk factor for stroke and myocardial infarction, represents one extreme of this trait. Intense research is currently on-going to identify genetic loci that regulate BP and predispose to hypertension. Such studies are being conducted in humans as well as in rodent models of genetic hypertension.1 One of the main functions of animal studies is to identify loci that can then be studied in human hypertension. However, for several loci that have been identified in this way,2 3 4 5 studies in humans have so far been negative.6 7 8 9 10 11 12 This may reflect species differences or the genetic heterogeneity of human hypertension and the complexities of studying multifactorial traits in outbred human populations. On the other hand, a recent study has provided strong direct evidence of the involvement of the angiotensinogen locus in human hypertension. In this study, Jeunemaitre et al13 showed an excessive sharing of angiotensinogen alleles in affected hypertensive sibships as well as an association of specific molecular variants of angiotensinogen with the trait. They estimated that mutations at the angiotensinogen locus may be a predisposing factor in 3% to 6% of hypertensive individuals younger than 60 years of age. The same molecular variants at the locus that were associated with hypertension were also found to be associated with increased plasma angiotensinogen levels. Two subsequent studies14 15 have provided independent evidence for the involvement of angiotensinogen in hypertension although a negative association study has also been reported.16 Angiotensinogen is the substrate in the renin-angiotensin cascade. Cleavage of the amino-terminal segment of angiotensinogen by renin releases a decapeptide, angiotensin I (Ang I), which is further processed by angiotensin-converting enzyme to produce the octapeptide Ang II, the active hormone of the cascade. Through actions on several organs, Ang II plays a central role in salt and water homeostasis and in the maintenance of BP.17
Given the above findings, our purpose in this study was to investigate the role of angiotensinogen in the elevated BP of the spontaneously hypertensive rat (SHR), the most widely studied genetic model of human essential hypertension. We analyzed the involvement of the angiotensinogen locus as a genetic determinant of BP in this model by linkage analysis in a large cohort of F2 rats derived from a cross of the SHR with the normotensive Wistar-Kyoto rat (WKY). We observed a significant effect of the locus on pulse pressure (systolic BP minus diastolic BP). In further studies, we compared the plasma levels and primary structures of SHR and WKY angiotensinogens and analyzed angiotensinogen expression in several tissues to identify possible mechanisms underlying this effect.
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Methods |
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Rats and Cross-breeding
SHR and WKY were obtained from colonies maintained by strict brother-sister mating in the Biomedical Services Unit, University of Leicester. The colonies were initially started from SHR and WKY breeding pairs derived from Charles River Laboratories (Margate, UK) at the same time as rats for cross-breeding (see below).
The generation and characterization of the F2 rats used in this study have been described in detail previously.18 Briefly, 6 SHR and 6 WKY (3 males and 3 females of each) derived from the breeding stock of Charles River Laboratories were mated to obtain F1 hybrids. F1 rats (16 males and 16 females) were randomly mated to generate 400 male F2 rats. Rats were housed under controlled conditions (temperature, 21±1°C; humidity, 60±10%; 12-hour day/night cycle). The first 233 F2 rats were fed standard rat chow (Rat & Mouse No. 3 Breeding Diet, Special Diet Services Ltd) containing 0.25% sodium and 0.66% potassium and given free access to tap water. The remaining 167 F2 rats were given the same diet as above but had 1% NaCl added to the drinking water for 10 weeks from 16 weeks of age until the termination of the study at 26 weeks of age.
BP Measurements in F2 Rats
Indirect systolic BP in the tail artery was measured by tail
plethysmography18 at 12, 16, and 20 weeks of age in rats
on a normal diet and at 16 (presalt), 18, and 20 weeks of age in rats
on a high salt diet. In addition, in 193 and 154 of the rats on normal
and high salt diets, respectively, direct BP was measured at 25/26
weeks of age with the use of a computerized
technique.19 20 Briefly, with rats under halothane
anesthesia (2% in oxygen), a catheter was inserted via the femoral
artery into the lower abdominal aorta, and the rat was placed into an
individual recording cage and allowed to recover for 24 hours. The
arterial catheter was connected to a BP transducer (Statham P23ID,
Gould Inc) via a rotating swivel that allowed the rats to move freely.
BP measurements were recorded beat-by-beat for 2 consecutive hours
between 10 AM and 5 PM. Data were processed
off-line as previously described.19 The whole recording
system was validated carefully in terms of signal dampening and
resonance phenomena in the catheter. With the use of a technique to
generate pressure-step functions,21 the system was shown
to be underdamped. The resonance frequency obtained after seven trials
was 33±0.4 Hz and the dampening factor was 0.14±0.03. Therefore, the
frequency response was higher than the frequencies usually observed in
pressure signals from rats, and the dampening factor was largely below
the critical dampening value.21 Calibration of the
instrument was verified before each measurement. All procedures were
carried out in accordance with our institutional guidelines.
Southern Blot Analysis
DNA was prepared from tail fragments and restriction enzyme
digests, and Southern blot analysis was carried out using standard
protocols.22 Probes used for this analysis were either
an almost full-length (1.64-kb) rat angiotensinogen cDNA (kindly
provided by Dr Kevin Lynch, University of Virginia, Charlottesville) or
subfragments of this cDNA (see "Results"). Filters were washed
under conditions of high stringency (0.1x SSC at 65°C). Molecular
weights of fragments were estimated from concurrently run aliquots of
the BRL 1-kb DNA ladder (Life Technologies).
Cloning and Sequencing of SHR and WKY Angiotensinogen Coding
Regions
Two micrograms of liver RNA from SHR and WKY prepared as
described below were reverse-transcribed using
oligo(dT)12-18 and then amplified22 using
angiotensinogen-specific primers. The upstream primer (AOG1) was
located in the 5' untranslated region of exon 1 (nucleotides 98-117,
sequence 5'-CTGTGCTTGTCTGGGCTGGA-3') and the downstream primer (AOG2)
located in the 3' noncoding region (nucleotides 1591-1572, sequence
5'-TGGAAGGAGTGACGGGAAGC-3'). The amplified products were then cloned
into M13 using restriction sites incorporated into the ends of the
primers and sequenced using angiotensinogen-specific primers designed
from the published cDNA sequence.23 To exclude polymerase
chain reaction artifacts, we verified point mutations by analysis
of several templates.
Northern Blot Analysis
Total RNAs were prepared by LiCl/urea
precipitation,24 and 60-µg aliquots were analyzed using
standard Northern blot techniques.22 The probes used were
the angiotensinogen cDNA described above and a 29-mer oligonucleotide
complementary to GAPDH mRNA25 as an internal control. Band
intensities were quantified by densitometry using an LKB 2222-010
Ultrascan XL scanner (LKB-Produkter AB).
Measurement of Plasma Angiotensinogen and Renin Concentrations
After rats were killed by decapitation, 1 mL of blood was
collected into prechilled tubes containing heparin. Plasma was
separated after being spun in a refrigerated centrifuge and was stored
at -80°C before analysis.
For measurement of plasma angiotensinogen concentration, duplicate plasma samples (10 µL) were mixed with mouse submaxillary gland renin (20 µL, 112 ng) and 30 µL of 1 mol/L TES buffer (pH 7.2) containing EDTA (50 mmol/L), 2,3-dimercaptopropanol (8 mmol/L), and phenylmethylsulfonyl fluoride (PMSF) (0.5 mg/mL) for 0, 60, and 90 minutes at 37°C, the last time point to ensure that the reaction had reached a plateau. Reactions were stopped by chilling the sample and adding 300 µL ice-cold 0.1 mol/L Tris-acetate, pH 7.4, containing human serum albumin (1 mg/mL). After dilution, the Ang I generated was measured by radioimmunoassay as described by Menard and Catt.26 Plasma angiotensinogen concentration was calculated from the 90-minute results assuming that 1 mol angiotensinogen generates 1 mol Ang I.
For measurement of plasma renin concentration, plasma samples (20 µL) were incubated with rat renin–free plasma (50 µL) containing 0.5 mg/mL PMSF and 0.1 mol/L Tris-acetate buffer (pH 7.4) containing 16 mmol/L 2,3-dimercaptopropanol (10 µL) for 120 minutes at 37°C. Rat renin–free plasma was prepared from rats binephrectomized 24 hours previously. The reactions were stopped by chilling the sample and adding 400 µL ice-cold 0.1 mol/L Tris-acetate, pH 7.4, containing human serum albumin (1 mg/mL). Subsequent determination of the Ang I generated was carried out as described above.
Statistical Analysis
Statistical analysis was carried out using
MINITAB Release 7 (Minitab Inc). Group means were
compared by one-way and two-way ANOVA. Interactions were analyzed using
general linear modeling.
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Results |
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Restriction Fragment Length Polymorphism at the Rat Angiotensinogen Locus
SHR and WKY DNAs were digested with 39 restriction enzymes and analyzed by Southern blotting with the full-length angiotensinogen cDNA. Polymorphisms between the DNAs were found with four enzymes only (Pst I, Pvu II, Nco I, and Acc I). The polymorphism detected with Pst I shown in Fig 1 was used to type F2 rats for their genotype at the angiotensinogen locus.
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Effect of Angiotensinogen Genotype on BP Phenotypes in
F2 Rats
Angiotensinogen genotype had no effect either on the
indirect BP values measured at 12, 16, and 20 weeks in the rats on the
normal salt diet or on any of the indirect BP values measured in the
rats placed on the high salt diet (data not shown). Direct BP values of
the rats of the two studies measured at 25/26 weeks of age grouped
according to their angiotensinogen genotypes are shown in the
Table. Again, no effect of genotype was seen on
either systolic or diastolic pressure in either study. However,
angiotensinogen genotype had a marginal (P=.07)
effect on pulse pressure in rats on the normal salt diet and a more
significant effect (P=.03) in rats on the high salt diet
(Table). To analyze the effect of genotype in the entire
population of F2 rats, we carried out a two-way ANOVA using
genotype and type of diet as the variables. In this
analysis combining the data of the two cohorts, the effect of
genotype on pulse pressure was highly significant (F=5.76,
P=.003). The effect of the diet, as expected, was also very
significant (F=33.13, P<.001) (Table), but there was no
interaction between diet and genotype (F=0.41,
P=.66). There was also no interaction of angiotensinogen
genotype and salt treatment for systolic and diastolic
pressures (F=0.09, P=.59 and F=0.81, P=.92,
respectively) (Table).
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Sequence Comparison of SHR and WKY Angiotensinogen Coding
Regions
To identify any differences in the primary structure of SHR and
WKY angiotensinogens, we sequenced the entire 1434 bp of SHR and WKY
angiotensinogen coding regions. Four differences were observed between
SHR and WKY cDNAs: at nucleotide 503: SHR (C), WKY (T); nucleotide 835:
SHR (G), WKY (A); nucleotide 976: SHR (A), WKY (G); and nucleotide
1247: SHR (C), WKY (T). None of the differences, however, result in an
amino acid substitution. At position 503, the SHR sequence corresponds
to the published Wistar sequence,23 and in the other three
cases, the WKY sequence corresponds to the published sequence.
The C
T change between SHR and WKY at position 503, which is located
in exon 2,27 causes the deletion of a Pst I
site in the latter. In addition to this, there is a further
Pst I site 78 bp downstream in the same exon common to SHR
and WKY. Since this was approximately the difference in size seen
between the polymorphic SHR and WKY Pst I fragments (Fig 1), to confirm position 503 as the location of the Pst I
polymorphism, we probed SHR and WKY DNAs digested with
Pst I with restriction fragments of the full-length
angiotensinogen cDNA. Three contiguous nonoverlapping fragments were
used: an EcoRI-BamHI fragment (approximately 500
bp) containing sequences in exon 1 and the 5' end of exon 2 (exon 1-2
probe); a BamHI-BamHI fragment (approximately 700
bp) containing sequences from the 3' end of exon 2 to the 5' end of
exon 4 (exon 2-4 probe); and a BamHI-HindIII
fragment (approximately 500 bp) containing a sequence from the 3' end
of exon 4 and a sequence from exon 5 (exon 4-5 probe). The
polymorphic bands (a/a' in Fig 1) were detected only by the exon
1-2 probe. Bands b and d were detected only by the exon 2-4 probe and
band c by the exon 4-5 probe.
Plasma Angiotensinogen and Renin Concentrations
To see whether there were differences in plasma angiotensinogen
concentration between SHR and WKY, we measured plasma angiotensinogen
concentration and, in addition, plasma renin concentration in
25-week-old SHR and WKY maintained on a normal salt or high salt diet
from 16 to 25 weeks of age, as per the F2 rats. The results
are shown in Fig 2. There was no strain (F=0.01,
P=.958) or diet (F=1.94, P=.17) effect on plasma
angiotensinogen concentration (Fig 2). On the other hand, plasma renin
concentration was significantly lower in SHR compared with WKY
(F=14.45, P<.001). Furthermore, the high salt diet
significantly reduced plasma renin concentration in WKY
(P=.005) but not in SHR (P=.52) (Fig 2).
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Angiotensinogen mRNA Levels in SHR and WKY Tissues
To see whether there were differences in angiotensinogen gene
expression in tissues of cardiovascular relevance between SHR and WKY,
we compared angiotensinogen mRNA levels in the liver, brain, kidney,
and aorta of (1) 6-week-old SHR and WKY, (2) 25-week-old SHR and WKY,
and (3) 25-week-old SHR and WKY treated with a high salt diet (1% NaCl
in drinking water) from 16 to 25 weeks of age.
Fig 3 shows angiotensinogen mRNA levels in the liver, brain, and kidneys of 6-week-old SHR and WKY. Levels did not differ in any of the tissues. At 25 weeks of age, angiotensinogen mRNA levels did not differ between SHR and WKY in the brain, but in both liver (F=23.3, P<.0001) and kidney (F=23.6, P<.0001), levels were slightly (25% to 70%) but significantly higher in WKY (Figs 4 and 5; compare lanes 1 through 8 and 9 through 16 in Fig 4). Salt treatment from 16 to 25 weeks of age by itself did not have a significant effect on angiotensinogen mRNA levels in any of the tissues.
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In the aorta, in contrast to the above tissues, a sixfold to sevenfold higher level of angiotensinogen mRNA was seen in 25-week-old WKY compared with SHR (Fig 6). Unlike other tissues, the level was also higher (1.7-fold) at 6 weeks in WKY. To confirm the striking difference in aortic angiotensinogen mRNA levels in adult SHR and WKY, we subsequently studied pooled aortas from a further six SHR and six WKY. An almost identical difference to that shown in Fig 6 was observed (data not shown).
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Discussion |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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In this article we present an analysis of the role played by angiotensinogen, the substrate of the renin-angiotensin cascade, in the elevated BP of SHR. We found that the SHR angiotensinogen locus cosegregated with a significant increase in pulse pressure but, surprisingly, not systolic and diastolic pressures individually in F2 rats derived from a cross of SHR with WKY. Not only the overall strength of the association (P=.003) but also the consistency of the observation in two rat cohorts derived sequentially from the cross and maintained on different diets add to the confidence in the data. However, it has recently been recommended that stricter criteria (P<10-4) should be used to deduce linkage in cosegregation studies,28 so our finding should be considered to be provisional until confirmation is obtained in further studies.
Although the absolute differences in pulse pressure between genotypes were relatively small for both cohorts (Table), these values need to be considered in the context of the spread of pulse pressure values, which were only 39 and 40 mm Hg in the normal and high salt cohorts, respectively. Calculation of the degree of genetic determination (ratio of genetic to total variance) of pulse pressure in the F2 rats as described by Rapp,29 using data from identically studied SHR and WKY as estimates for the environmental variance, gave values of 45% and 57%, respectively, for the two cohorts. Thus in relative terms, the locus accounted for approximately 20% of the genetic (10% of the total) variance in pulse pressure in the F2 rats. In addition, since pulse pressure varies with age30 and we only measured it at one time point (25/26 weeks), it is possible that both the absolute and relative effects of the angiotensinogen locus on pulse pressure are greater at other ages. It also needs to be emphasized that the effect of the locus may be cross specific, as in a cross between stroke-prone SHR and WKY, no effect was seen.31
Because pulse pressure is the difference between systolic and diastolic pressures, it is not a completely independent variable. However, of the factors that influence systolic and diastolic pressures, only those that have a differential effect on these two parameters will alter pulse pressure. Hemodynamically, three mechanisms in particular have been identified as causing an increase in pulse pressure30 : (1) an increase in the volume or velocity of ventricular ejection, (2) a modification of the timing of reflected waves, and (3) a reduction in the viscoelastic properties (compliance) of the arterial wall. The first two of these will predominantly affect systolic pressure, whereas a decrease in compliance will tend to increase systolic and decrease diastolic pressure. The trends in systolic and diastolic pressures in our rats (Table) suggest that this mechanism, ie, a decrease in compliance of the arterial tree, is the most likely explanation for the association of the angiotensinogen locus with pulse pressure and probably explains why significant effects on systolic and diastolic pressures individually were not seen. It also needs to be emphasized that unlike mean arterial pressure, pulse pressure varies through the arterial tree.30 We measured the pressure in the lower aorta, and thus to be more specific, the association we observed can only be related to measurements made at this site.
Our finding supports the concept that different loci may influence different hemodynamic components of BP and that pulse pressure has specific genetic determinants.32 In a previous study by Dubay et al,32 a locus on chromosome 2 was found to cosegregate with pulse pressure in a cross of Lyon hypertensive and Lyon normotensive rats. The location of the angiotensinogen gene on rat chromosome 19 therefore identifies a novel locus influencing pulse pressure. The significance of such findings relates to the increasing recognition of pulse pressure as an independent determinant of cardiovascular risk in hypertension.30
Our cosegregation data identify an effect on pulse pressure of the angiotensinogen locus but do not necessarily implicate the angiotensinogen gene. The effect could be due to a linked gene. Depending on its distance from the angiotensinogen gene, the true effect of the locus on pulse pressure could be much greater. However, given the widespread cardiovascular effects of Ang II, for which angiotensinogen is the only precursor, the angiotensinogen gene is an obvious candidate gene. To identify possible mechanisms by which the angiotensinogen gene might influence pulse pressure, we examined the primary structure of angiotensinogen in SHR and WKY and compared plasma levels and the levels of expression of the gene in several tissues in the two strains. With regard to the former, a number of differences were observed in the coding regions of SHR and WKY transcripts, but none of these resulted in amino acid substitutions. Thus, these data indicate that the effect of the locus on pulse pressure is not mediated through any structural difference in the angiotensinogens produced by the two strains. Similarly, we found no difference in the level of circulating angiotensinogen between 25-week-old SHR and WKY on either a normal or high salt diet (Fig 2), a finding that may seem paradoxical given the higher level of angiotensinogen mRNA present in WKY liver compared with SHR liver (Figs 4 and 5). However, there was also a difference in plasma renin concentrations between the strains (Fig 2); therefore, a relatively higher consumption of angiotensinogen in WKY may at least partly account for the similar plasma levels of substrate. In addition, differences in peripheral metabolism of angiotensinogen between the strains cannot be excluded.
The angiotensinogen gene is expressed in a wide range of tissues in addition to the liver, including the brain, kidney, and aorta, which influence cardiovascular function.33 Therefore, we also examined angiotensinogen mRNA levels in these tissue sites and the liver in 6- and 25-week-old SHR and WKY. We studied young rats (6 weeks) in addition to the adult animals to try to exclude secondary effects of hypertension on angiotensinogen gene expression. At this age, the indirect BP values of the rats were not significantly different (SHR, 100.0±2.9 mm Hg; WKY, 97.5±1.9 mm Hg). Our findings from these studies demonstrate tissue-specific, age-related differences in angiotensinogen mRNA levels between SHR and WKY. Angiotensinogen mRNA levels did not differ in the liver, brain, or kidney at 6 weeks of age or in the brain at 25 weeks of age. However, in the liver and kidney, the levels at 25 weeks were slightly but significantly higher in WKY in both cases (Figs 4 and 5). Pratt et al34 have previously reported a similar difference in kidney angiotensinogen mRNA levels in 12-week-old SHR and WKY, although at this age they did not find any differences in hepatic angiotensinogen mRNA levels. Interestingly, they found that sodium depletion further increased kidney angiotensinogen mRNA level in WKY but not in SHR.
Somewhat in contrast to the findings in the above tissues, a much more striking difference in angiotensinogen mRNA level was seen in the aorta. At both 6 and 25 weeks of age, WKY had higher aortic angiotensinogen mRNA levels than SHR (Fig 6). These findings need to be interpreted with some caution because they are based on RNA extracted from pooled aortas (pooling was necessary because of the limited amounts of tissue available from individual rats). However, the consistency of the observation at two ages, our finding of an almost identical result in aortas taken from further groups of adult SHR and WKY several months after the original study, and the fact that a difference of a similar magnitude has been reported previously in adult SHR and WKY35 suggest that there is a substantial difference in angiotensinogen gene expression in the aortas of SHR and WKY that is present from an early age.
Various mechanisms can be postulated to explain the differences observed in angiotensinogen gene expression between SHR and WKY. The finding that levels are already different in the aortas of 6-week-old rats suggests that the difference, in this tissue at least, is not entirely a secondary consequence of a rise in BP. However, our BP measurements in rats at this age were indirect, and a subtle difference cannot be excluded. Certainly in the adult SHR, one cannot exclude a hemodynamic factor suppressing angiotensinogen gene expression in this site as well as in the kidney. Second, it is well recognized that Ang II is an important regulator of hepatic angiotensinogen production.36 Thus, the lower plasma renin concentration found in the 25-week-old SHR (in itself a likely consequence of the hypertension) could lead to a decrease in angiotensinogen mRNA levels, at least in this and possibly some other sites, through a reduction in circulating Ang II. Finally, in the context of our cosegregation data, an important mechanism to consider is that there may be a primary difference in the regulatory regions of the SHR and WKY angiotensinogen genes through which trans-acting factors cause tissue-specific and age-specific differences in gene expression in the two strains. This explanation would gain support if further studies found that the angiotensinogen genotype also cosegregated with angiotensinogen mRNA levels in F2 rats.
Given the fact that the probable mechanism by which allelic variants at the angiotensinogen locus influenced pulse pressure in our F2 rats was through an effect on aortic compliance (see above), the finding of a lower aortic angiotensinogen mRNA level in SHR compared with WKY is interesting although somewhat contrary to expectation. Aortic angiotensinogen gene expression has been localized to both the adventitia and periaortic adipose tissue as well as the medial smooth muscle cell layer.37 38 In a previous study35 that compared aortic angiotensinogen mRNA levels in SHR and WKY, the increased angiotensinogen mRNA level in WKY was localized to aortas stripped of endothelium and adventitia. In the present study, periaortic fat was removed although no attempt was made to separate the adventitia. Ang II has been shown to have multiple effects on aortic wall structure and function independent of any effects mediated through BP elevation. These include effects on vascular smooth muscle cell growth, extracellular matrix formation, sympathetic nervous system activity, and endothelial function.39 The general consensus is that such effects, particularly those on vascular structure, would tend to increase the rigidity of the vascular wall. If angiotensinogen formed in the aortic wall is a determinant of the level of locally generated Ang II, one might anticipate, given our findings, that any effect of such Ang II on reducing compliance would be greater in WKY than in SHR. In support of this, a recent study by Levy et al40 showed that directly measured aortic compliance was increased to a much greater extent in WKY than in SHR by 12 weeks of treatment with the angiotensin-converting enzyme inhibitor perindopril. In contrast, as an illustration of the complex factors that regulate compliance, they found that reduction in media thickness was more pronounced in SHR. Thus, while our findings on aortic angiotensinogen mRNA levels seem paradoxical and difficult to easily reconcile with the cosegregation data, the complex nature of both pulse pressure and aortic compliance as phenotypes and the poorly understood role or roles of angiotensinogen gene expression in extrahepatic sites require an open-minded interpretation of the data.
Finally, how do our findings relate to the observations that have been made in human hypertension? Subjects in the study by Jeunemaitre et al13 were primarily identified on the basis of a diagnosis of essential hypertension requiring antihypertensive treatment before the age of 60. Since the majority of subjects were on antihypertensive medication at the time of enrollment in the study, analyzable BP data were not available. Thus it is difficult to directly compare their findings with ours. However, since the criterion by which subjects in their populations were classified as being hypertensive was the usual one of an elevation of diastolic (±systolic) pressure, it is possible that the locus affects different BP phenotypes in humans and rats. Furthermore, from a mechanistic point of view, and in contrast to our finding, they also observed an effect of the angiotensinogen genotype on plasma angiotensinogen levels.13 Thus, the findings from SHR and humans would appear to differ. However, our results suggest that it would be worthwhile in future studies in humans to examine the effects of the locus on individual BP parameters in subjects not receiving antihypertensive medication.
In conclusion, we have observed a small but specific effect of the angiotensinogen gene locus on pulse pressure in F2 rats derived from a cross of SHR and WKY. This effect is not caused by a modification of the angiotensinogen protein or a difference in plasma angiotensinogen levels in adult rats of the two strains but may be related to a tissue-specific abnormality of angiotensinogen gene expression in SHR aorta. However, further studies are needed both to confirm the specific effect of the locus on pulse pressure and to definitively identify the responsible gene at the locus and the mechanism through which it influences this important cardiovascular parameter.
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Acknowledgments |
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We gratefully acknowledge support from the British Heart Foundation, the National Kidney Research Fund, the EURHYPGEN Concerted Action of the European Commission, and the Centre National de la Recherche Scientifique. We are grateful to Dr Kevin Lynch for providing the angiotensinogen cDNA. We thank Valerie Orea, Pascale Privat, Dr Derek Forbes, and the staff of the Biomedical Services Unit, Leicester University, for their technical assistance.
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Footnotes |
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Reprint requests to Dr N.J. Samani, Department of Medicine, University of Leicester, Clinical Sciences Building, Leicester Royal Infirmary, PO Box 65, Leicester LE2 7LX, UK.
Received June 3, 1994; first decision July 18, 1994; accepted January 12, 1995.
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