A quantitative trait locus for aortic smooth muscle cell number acting independently of blood pressure: implicating the angiotensin receptor AT1B gene as a candidate

Julie Dutil, Vasiliki Eliopoulos, Éve-Lyne Marchand, Alison M. Devlin, Johanne Tremblay, Kalyani Prithiviraj, Pavel Hamet, Annik Migneault, Denis deBlois and Alan Y. Deng

Research Centre-Centre Hospitalier de l’Université de Montréal (CHUM), Montreal, Quebec, Canada


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Vascular hyperplasia may be involved in the remodeling of vasculature. It was unknown whether there were genetic determinants for aortic smooth muscle cell number (SMCN) and, if so, whether they acted independently of those for blood pressure (BP). To unravel this issue, we utilized congenic strains previously constructed for BP studies. These strains were made by replacing various chromosome 2 segments of the Dahl salt-sensitive (S) rat with those of the Milan normotensive rat (MNS). We measured and compared SMCN in aortic cross-sectional areas and BPs of these strains. Consequently, a quantitative trait locus (QTL) for SMCN was localized to a chromosome region not containing a BP QTL, but harboring the locus for the angiotensin II receptor AT1B (Agtr1b). Agtr1b became a candidate for the SMCN QTL because 1) two significant mutations were found in the coding region between S and all congenic strains possessing the MNS alleles, and 2) contractile responses to angiotensin II were significantly and selectively reduced in congenic rats harboring the MNS alleles of the SMCN QTL compared with S rats. The current investigation presents the first line of evidence that a QTL for aortic SMCN exists, and it acts independently of QTLs for BP. The relevant congenic strains developed therein potentially provide novel mammalian models for the studies of vascular remodeling disorders.

functional genomics; angiotensin II receptor AT1B; aortic hyperplasia; vasoreactivity; congenic strains


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
HYPERTROPHY AND/OR VASCULAR HYPERPLASIA may contribute to the elasticity/rigidity of the blood vessel and peripheral vascular resistance. Although cardiac hypertrophy and hypertension were sometimes associated, there were indications that genes for blood pressure (BP) and left ventricular hypertrophy (LVH) could be separated in hypertensive models (3, 22). LVH can be determined by genes from normotensive rats independently of hypertension (28).

As for a relationship between hypertension and hyperplasia, the initial evidence indicated that the cultured aortic smooth muscle cells (SMC) proliferated faster (i.e., hyperplasia) in the spontaneously hypertensive rats (SHR) than in the Wistar Kyoto rats (WKY) (21, 41). These findings suggested a link between hypertension and the SMC proliferation. Since then, a large body of evidence has been accumulated on their associations (5, 14, 21, 23, 27, 38). On the basis of these observations, one would expect that a higher SMC proliferation might correlate with a higher BP. Nevertheless, there was one genetic study reporting that hypertension was not associated with the growth of skin fibroblast in a F2 cross between SHR and WKY (20). This is not surprising, because an association between BP and the SMC growth in these two strains could simply be accidental, not causal, one way or the other.

To date, it has remained unclear whether there are quantitative train loci (QTLs) determining vascular SMC number and, if so, whether mechanisms determining hypertension and vascular hyperplasia could be separated. Our current goal is to discover QTLs controlling vascular SMC number regardless of, or unprejudiced by, association or dissociation with BP.

The power and utility of genetic analyses are evident in finding causal genes for vascular hyperplasia. The beauty of the genetic approach lies in its objectivity, i.e., one is not biased toward predicting an outcome based on a preconceived notion linking two associated phenomena, e.g., hypertension and hyperplasia. Nor are we biased toward the perception that hyperplasic alleles of a QTL, if it existed, had to come from a hypertensive strain, not from a normotensive strain. If hypertensive alleles of a QTL for BP could originate from a normotensive strain, and vice versa (2, 31), one could expect that SMC-increasing alleles of a QTL could originate from a normotensive strain.

Ever since the revelation of a QTL for BP in Dahl salt-sensitive (S) rats (9, 13), rat chromosome (Chr) 2 appears to play an important role in the development of hypertension in several of the hypertensive strains (1, 10, 11, 15, 18, 24, 33, 34, 36, 39, 40). In the process, a number of congenic strains have been developed that cover various regions of Chr 2 of S rats (16, 17). Taking advantage of these resources, some questions could be addressed regarding QTLs for hyperplasia. Specifically, 1) is there a QTL for vascular hyperplasia and particularly for aortic smooth muscle cell number (SMCN) in S rats? If so, does it act independently of blood pressure? And 2) if there is a QTL specific for SMCN, can any gene be supported as a candidate?

In the present studies, BPs of certain congenic strains were compared along with their respective indexes of vascular SMCN, vascular, cardiac, and renal weights, and internucleosomal DNA fragmentation, an indicator of apoptosis. In addition, when a candidate gene was found in the region containing the QTL for SMCN, sequencing would be conducted to detect significant mutations that could have the potential to alter the expression or/and function of the gene.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals
For the sake of easy distinctions for congenic strains specifically made for Chr 2, a prefix of C2 is added in front of congenic designations. Thus the congenic strains previously utilized (16, 17), S.M, S.M1, S.M2, and S.M6 are redesignated C2S.M, C2S.M1, C2S.M2, and C2S.M6, respectively (Fig. 1).



View larger version (29K):
[in this window]
[in a new window]
 
Fig. 1. Mapping of a quantitative trait locus (QTL) for vascular smooth muscle cell no. (SMCN) independent of blood pressure (BP) QTLs. The linkage map is essentially the same as that published previously (913) and is based on an F2 [Dahl salt-sensitive rat (S) x Milan normotensive rat (MNS)] population. Nos. to the left of the linkage map are units in cM. RH map refers to the map using rat/hamster radiation hybrids, in which units are in cR. Nos. on the RH map are arbitrary units descending from the top of the chromosome. Solid bars under congenic strains symbolize the S chromosome fragments that have been replaced by those of the MNS rat. The entire region indicated by solid bars and junctions between the solid and open bars are homozygous for MNS, i.e., MM, on the map for all the markers listed in the corresponding positions. Open bars on ends of solid bars indicate the ambiguities of crossover breakpoints between markers. Adh, alcohol dehydrogenase; Agtr1b, angiotensin receptor type-1B; Atp1a1, Na+-K+-ATPase-{alpha}1; Camk2d, calmodulin-dependent protein kinase II{delta}; Fgg, fibrinogen-{gamma}; Gca, guanylyl cyclase A/atrial natriuretic peptide receptor; Mme, membrane metallo-endopeptidase (neutral endopeptidase, enkephalinase); Prlr, prolactin receptor. D2Chm90 is a newly produced marker. Primers used are forward 5'-TTGGAGTTCATTAAGCAACACAG-3' and reverse 5'-CCTTCTGGAAAAA-GGTAAACCA-3'. The rest of the markers are anonymous. Congenic strains were as follows: C2S.M, S.MNS-(D2Mit6-Adh)/Lt; C2S.M1, S.MNS-(D2Mit6-D2Rat166)/Lt; C2S.M2, S.MNS-(D2Mit6-D2Rat303)/Lt; and C2S.M6, S.MNS-(Mme-D2Rat131)/Lt. MAP refers to the averaged mean arterial pressure during the period of measurement for each strain (16, 17). ANOVA with Dunnett’s correction compares MAPs between S and each of the congenic strains. The placement of the QTL specifically for SMCN is indicated by a bracket, as is done for BP C2QTL1 and BP C2QTL2.

 
Marker Development
We examined all the available markers in the rat database for the region between D2Rat166 and Mme (Fig. 1). But none of them was either homozygous SS or MM for C2S.M1 and C2S.M6. Therefore, potential overlaps could not be ruled out between the lower segment for BP C2QTL1 (17) and the upper segment for BP C2QTL2 (16). This ambiguity called into question whether or not BP C2QTL1 and BP C2QTL2 were separate or merely represented the same QTL, but under the influence of a different background.

To prove that BP C2QTL1 and BP C2QTL2 are separate genes, one would need to find a marker between D2Rat166 and Mme that is SS for both C2S.M1 and C2S.M6. For this purpose, we employed the rat genome database. Supercontigs located in the Chr 2 region between D2Rat166 and Mme were first identified by blasting them at the website http://www.ncbi.nlm.nih.gov/genome/seq/RnBlast.html. From such a supercontig, regions containing microsatellites were searched and, when found, were used to design markers for genotyping rats based on PCR. These new markers were designated with D2Chm prefixes, which represent CHUM. Consequently, a series of markers were generated. One particular marker designated D2Chm90 was polymorphic and located in the region of interest (Fig. 1).

BP Studies and Tissue Extractions
BP studies on the congenic strains were reported in detail previously (16, 17). In brief, male rats were weaned at 21 days of age, maintained on a low-salt diet (0.2% NaCl; Harlan Teklad 7034), and then fed a high-salt diet (2% NaCl; Harlan Teklad 94217) starting from 35 days of age until the end of the experiment. Telemetry probes were implanted when rats were 56 days old (i.e., after 3 wk of the high-salt diet), with their body weights between 250 and 320 g. The BP measurements lasted until the time of death.

Rats were killed by decapitation 30 days after the commencement of their BP measurements, i.e., at 14 wk of age. The organs of interest (see below) were removed, cleaned carefully from surrounding adventitial connective tissues and fat, blotted to remove excess blood, and weighed immediately. To minimize any potential inconsistency in the collection, one person was designated for harvesting one particular organ and also for the subsequent dissections into subsections of interest.

The whole heart was then dissected into the left ventricle plus the septum, and into the right ventricle. The weight of each section was then recorded and corrected for the body weight of the respective animal. The dissection was performed according to a previously published procedure (29, 37).

Measurement of Aortic Cross-Sectional Areas
The following procedure was done, essentially according to a previously published protocol (5). The thoracic aorta was isolated from the diaphragm to above the first intercostal artery and cleaned of adherent adventitial tissue and fat. A 3-mm-long ring of aorta was cut between the third and fourth intercostal arteries. The aortic rings were immerse fixed in 4% parformaldehyde overnight and processed according to routine histological procedures for morphological examinations in paraffin-embedded tissues. The fixation of aortas was done in none-pressure-controlled conditions. The aortic endothelium of the remaining aorta segments was removed by scrubbing the intimal surface with a cotton-tipped applicator. The aortic media was immediately snap frozen in liquid nitrogen and kept at –80°C until further processing for DNA extraction.

The medial cross-sectional area was evaluated in 5-µm-thick, hematoxylin-eosine-stained sections of aorta. Photomicrographs of the aortic sections were taken at x400 magnification, digitalized, and analyzed using the public domain NIH Image program 1.61 (developed at the National Institutes of Health and available at http://rsb.info.nih.gov/nih-image/). Two independent measurements of each tissue section were conducted to ensure the reproducibility of the image analysis.

Determination of Vascular SMCN
The following was done essentially according to a procedure previously published (5). Briefly, three consecutive sections (5-µm thickness) were obtained from aortic rings sectioned perpendicularly to the longitudinal axis of the vessel. Tissue sections were stained with hematoxylin, photographed, and printed at a final magnification of x400. In the top section, an area, ad, of vessel wall was delineated by two parallel lines approximately perpendicular to the wall edges. A disector was defined as the three-dimensional probe bounded by ad and the top surface of the "bottom" section. The disector has volume vd = ad x hd, where ad is the area of the disector and hd is the height of the disector. The term hd is obtained by the following equation: hd = (s – l) x t, where s is the number of serial sections and t the average section thickness. Within ad, the number of nucleus profiles, nt, was determined, and in the subsequent sections, each of the nt nuclei was followed and marked. In the final section, the number of nuclei still present was determined, nb. The number nd = (nt – nb) is then the number of "downward-pointing" nucleus ends within the disector when counting from top to bottom. Binucleate SMC account for <0.5% of the SMC in the thoracic aorta of hypertensive rats (30). Therefore, on the assumption that each cell contains only one nucleus, an estimate of cell numerical density, Nv, is given by Nd = nd/vd. The number of cells per unit vessel length then was estimated from a1 x Nv, where a1 is the medial cross-sectional area determined with the image analyzer as described above.

DNA Content and Apoptosis
The remaining aortic media was pulverized in liquid nitrogen. The total tissue DNA was extracted and quantified as described previously (5). To quantify the degree of internucleosomal DNA fragmentation, a hallmark of apoptosis (programmed cell death), the 3'-OH ends in the extracted DNA were radiolabeled using [32P]dNTP and terminal deoxynucleotidyl transferase. DNA was separated by gel electrophoresis and blotted, and radioactivity incorporated in the small fragments (150–1,500 bps) was quantified using a PhosphoImager as described previously (5).

Vasoreactivity Studies
Vasoactive responses to angiotensin II (ANG II) and phenylephrine (PE) were examined as described previously (26) in rings of thoracic aorta isolated from S and C2S.M2 rats implanted or not with a telemetry probe. Briefly, rats were killed by exposure to CO2, and freshly isolated vessels without adventitial adipose and connective tissue or endothelium were cut into 3-mm-long rings to be placed at 37°C in organ chambers filled with oxygenated Krebs solution containing, in mM, 11 dextrose, 117.5 NaCl, 1.18 MgSO4, 1.2 KH2PO4, 25 NaHCO3, 4.7 KCl, and 2.5 CaCl2. Isometric contractions were measured with the use of isometric force transducers (Harvard Apparatus) and a digitalized data acquisition system (Model MP100, Biopac System; Harvard). Rings were equilibrated under a passive tension of 1 g and then challenged with 40 mmol/l KCl to document receptor-independent contractility. Tissues were also stimulated with cumulative concentrations of PE (1–100 mmol/l) and ANG II (300 pmol/l to 300 µmol/l). Separate sets of rings were used to measure contractile responses in the presence or absence of nitro-L-arginine methyl ester (L-NAME; 100 µmol/l). The integrity of the endothelium was evaluated on the plateau of PE-induced maximal contraction by measurement of the relaxant response to acetylcholine (Ach; 1 µmol/l). In a subset of tissues, ANG II-induced contractile effect was also evaluated in the presence of losartan (75 nmol/l).

Statistical Analysis
In BP analyses, repeated measures ANOVA, followed by Dunnett’s test (which permits a correction for multiple comparisons and sample sizes), was used to compare a parameter between two groups as presented previously (16, 17). During the comparison, ANOVA was first used to analyze the data to see whether there was any difference among the groups. If the difference was significant, then the Dunnett’s test followed, to see which group was different and the level of significance compared with the S strain. If the difference was not significant, then no Dunnett’s test followed. In functional vasoreactivity studies, data from S and C2S.M2 were compared by unpaired Student’s t-test using Prism 4.0 (GraphPad Software).


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Localization of a QTL for Vascular SMCN Independent of BP QTLs
The marker D2Chm90 is important (Fig. 1). It came from the same supercontig, NW_043524, containing Mme and is homozygous MM for C2S.M but SS for C2S.M1, C2S.M2, and C2S.M6 (Fig. 1). Thus D2Chm90 effectively separated C2S.M1 from C2S.M6.

The most informative strain is C2S.M2 (Fig. 1), which had no effect on BP (Ref. 17 and summarized in Fig. 1, bottom) and had the same cardiac, left ventricular, and renal weights as S (Fig. 2, A and B) but showed higher SMCN (Table 1). In fact, aortic SMC counts of both C2S.M1 and C2S.M2 were higher than that of the S strain (P < 0.039). In contrast, SMC counts of the C2S.M6 and C2S.M strains were not different from that of the S strain (Table 1).



View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2. Comparisons of cardiac and renal parameters and cross-sectional areas of aortas between the congenic and S strains. The no. of rats for each strain in the comparisons is as follows. A: C2S.M (n = 5), C2S.M1 (n = 9), C2S.M2 (n = 10), C2S.M6 (n = 5), and S (n = 11). B: C2S.M (n = 5), C2S.M1 (n = 5), C2S.M2 (n = 10), C2S.M6 (n = 5), and S (n = 7). C: C2S.M (n = 5), C2S.M1 (n = 8), C2S.M2 (n = 8), C2S.M6 (n = 4), and S (n = 10). HW, heart weight; LVW and RVW, left and right ventricular weights, respectively; KW, kidney weight; LKW and RKW, left and right kidney weights, respectively; BW, body weight; CSAs, cross-sectional areas of aortas. HW, LVW, RVW, KW, LKW, and RLW were corrected for BW. For congenic strain designations, see Fig. 1 legend.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Comparisons in SMCN between congenic and S strains

 
Figure 2C shows comparisons of aortic cross-sectional areas (CSAs) between a congenic and the S strain. C2S.M6 had smaller CSAs than S (P < 0.006). CSAs (Fig. 2C) showed the same tendency as the SMC counts (Table 1). Both C2S.M1 and C2S.M2 had slightly larger, although not significantly different, CSAs than S.

Figure 2 also shows comparisons of cardiac and renal weights corrected for body weights among the strains. Kidney weights in C2S.M1, C2S.M6, and C2S.M were lower (P < 0.03) than in S rats, whereas those of C2S.M2 were not different from those in S rats (Fig. 2, A and B). Cardiac and left ventricular weights were only significantly lowered in C2S.M and C2S.M6 (P < 0.03) but not in C2S.M1 strains (P > 0.10). Table 2 shows assessments of DNA content and DNA fragmentation indexes in both cardiac ventricles and aortas.


View this table:
[in this window]
[in a new window]
 
Table 2. Comparisons in cardiac and vascular parameters between congenic and S strains

 
In terms of the DNA fragmentation indexes and DNA contents, in comparing various strains, a possible relationship between apoptosis (or DNA synthesis) and hypertension or aortic SMC number remains to be investigated further. There was a suggestive correlation between tissue weights and the DNA content in both ventricles (Table 2). Moreover, although the DNA fragmentation suggested a tendency to be inversely correlated with the DNA content in the left ventricle, there was a significant decrease in DNA content in the C2S.M and C2S.M6 strains compared with the S strain. In the right ventricle, the DNA fragmentation was significantly increased in the C2S.M strain only.

Angiotensin Receptor AT1B (Agtr1b) Gene as a Candidate for the SMCN QTL
In previous pharmacological studies, we observed that AT1 and not AT2 receptors were involved in the SMC growth (5, 38), implying that AT1 receptors might be important in mediating the SMC proliferation. There are two types of AT1 receptors, AT1A and AT1B, encoded by Agtr1a and Agtr1b genes, respectively. In our congenic strains, the Agtr1a gene on Chr 17 (8) was the same as in S (10, 16, 17), but the Agtr1b on Chr 2 (Fig. 1) was different in that it contained alleles from the Milan normotensive strain (MNS; Fig. 1). Because it is located in the chromosome region harboring the SMCN QTL (Fig. 1), Agtr1b could be conceived as a candidate gene for the SMCN QTL. We reasoned that to qualify Agtr1b to be the candidate gene, it had to contain significant mutations that could potentially result in differences either in its function or in its level of expression between S and congenic strains trapping the SMCN QTL.

Mutational evidence supporting Agtr1b as a candidate gene of the SMCN QTL.
Indeed, when Agtr1b genes were cloned and sequenced, several nucleotide mutations were detected in the coding region. Two of these mutations were of special interest in that they were found in MNS, C2S.M, C2S.M1, and C2S.M2 but were different from those in C2S.M6 and S, which had the same nucleotides. These two nucleotides resulted in significant changes in amino acids. One T-to-C change at nucleotide position 6 caused a change of (aliphatic) isoleucine to (hydroxyl) threonine at amino acid position 2. Another A-to-G change at nucleotide position 118 resulted in a significant change of (sulfuric) methionine to (aliphatic) valine at amino acid position 20. The nucleotide positions were numbered from the sequence coding for the first amino acid, as given previously (12). These mutations support functional implications of Agtr1b as the candidate for the SMCN QTL (Fig. 1), since C2S.M, C2S.M1, and C2S.M2 possess MNS alleles at Agtr1b (i.e., C at position 6 and G at position 118), whereas C2S.M6 possesses S alleles at Agtr1b (i.e., T at position 6 and A at position 118).

Functional evidence supporting Agtr1b as a candidate gene of the SMCN QTL.
If the mutations found in Agtr1b were truly significant, one would expect to observe functional differences between the MNS Agtr1b and S Agtr1b genes.

For the subsequent functional study, C2S.M2 was chosen because it carries a smaller chromosome interval of MNS than C2S.M1 (Fig. 1). In our work, contractile responses to ANG II were documented in rings of aorta isolated from C2S.M2 and S rats. The analysis indicated that contractile responses were not different, whether a telemetry probe had been implanted (n = 6/strain) or not (n = 6–7/strain), and the data were therefore pooled for each strain. As shown in Table 3, contractile responses to ANG II were significantly reduced in C2S.M2 rats compared with S rats, without a significant change in apparent receptor affinity. The contractions induced by ANG II were AT1 receptor dependent, since they were significantly inhibited (>90%) in the presence of losartan (data not shown). In contrast, there was no interstrain difference in the contractile responses to phenylephrine ({alpha}1-adrenergic agonist) or KCl (receptor-independent contractile stimulus) (Table 3). Similar conclusions were reached with tissues challenged in the presence or absence of the nitric oxide synthase inhibitor L-NAME (Table 3). Thus the hyperplasic aorta from C2S.M2 rats exhibited a selective decrease in ANG II-induced AT1 receptor-dependent contractility compared with S rats.


View this table:
[in this window]
[in a new window]
 
Table 3. Comparisons in contractile responses to KCl, angiotensin II, and phenylephrine between congenic strain C2S.M2 and S strain

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Major findings from the current studies are as follows. 1) There is a QTL(s) determining vascular SMCN that acts independently of QTLs for blood pressure. 2) The Agtr1b is a candidate for such a QTL based on two lines of evidence, gene mutations and their functional consequences. That is to say that significant nucleotide differences resulted in functional differences in Agtr1b between the S strain and the congenic strain carrying Agtr1b alleles from MNS. The significant functional correlates were shown in vasoreactivity studies.

A QTL for the Vascular SMCN Functions Independently of QTLs for Hypertension
C2S.M1 had a lower BP than S (Fig. 1 and Ref. 17) but had more aortic SMCN (Table 1). In contrast, C2S.M2 had the same BP as S (Fig. 1 and Ref. 17) but showed SMCN similar to those of C2S.M1 (Table 1). This fact clearly indicates that these genetic determinants for BP and aortic SMCN act independently of each other. On examination of the chromosome interval on the map, the QTL for the aortic SMCN should be present in the chromosome fragment shared between C2S.M1 and C2S.M2, which is between Prlr and D2Rat303 markers (Fig. 1). In contrast, BP C2QTL1 exists in the segment not shared between C2S.M1 and C2S.M2, i.e., between the D2Rat303 and Mme markers (Fig. 1). It is noted that the QTL for the vascular SMCN acts independently also of cardiac, left ventricular, and renal hypertrophy, because C2S.M2 had the same cardiac, left ventricular, and renal masses as S (Fig. 2, A and B).

Using two congenic strains, a stringent genetic testing, we found that aortic SMCN and BP have different genetic, and possibly physiological, bases. Although MNS rats per se were not directly analyzed for SMCN, C2S.M6 served effectively as a negative control, because it contained a small segment from the MNS strain not including the region delineated by Prlr and D2Rat303 markers (Fig. 1). Because C2S.M6 has no hyperplastic effect, whereas C2S.M1 and C2S.M2 do (Table 1), the effects to increase SMCN in C2S.M1 and C2S.M2 have to be attributed to the QTL residing in the Prlr/D2Rat303 segment, not to any MNS genetic background. The MNS rat would not be a good control, because it differs greatly from the S genome on the rest of the chromosomes (19, 25) and because it gives no information on the effects of genome changes during the congenic construction (7).

Because SMCNs were slightly different (Table 1) between C2S.M1 (170 ± 11) and C2S.M2 (134 ± 21), a possibility cannot be ruled out that there could be another QTL for SMCN in the region of BP C2QTL1 (Fig. 1). In that case, the same QTL could be responsible for both BP and SMCN.

It is noteworthy that the alleles to increase SMCN come from MNS, which is normotensive. Nonetheless, it is not surprising that MNS QTL alleles cause SMCN to increase, whereas S QTL alleles decreased SMCN. In an independent study, Lewis rats, a normotensive strain, actually carry BP-increasing alleles compared with S at a QTL on Chr 3, although the overall BP of the Lewis strain is considerably lower than that of the hypertensive S strain (31). This fact indicated that the BP-increasing QTL alleles in Lewis were hidden and were only unveiled by isolating the region harboring it from the rest of the Lewis genome by congenic strains. Therefore, our present and other studies further demonstrated the power and necessity of genetic dissections of individual QTLs using congenic strains.

The detection of BP C2QTL1 added further proof supporting the power of the congenic approach. In linkage analysis, there was no indication that a QTL existed in the region around C2QTL1 (9, 12). It is only after the isolation of a segment by use of a congenic strain (C2S.M1) that BP C2QTL1 was found (17). This phenomenon is due, probably, to epistatic gene-gene interactions (6, 7) among other things and indicates that the effect of the genetic background impacted by other QTLs affecting the same phenotype may need to be removed for the manifestation of a QTL for SMCN (Fig. 1).

It is also of note that the C2S.M strain included the chromosome segment contained in C2S.M1 and C2S.M2 (Fig. 1), and yet, C2S.M did show hyperplasia (Table 1). This phenomenon can be explained by the following. There could be another QTL or a modifying gene present in the segment in C2S.M that is lacking in C2S.M1, i.e., between D2Rat166 and Adh, that would have a hypoplasic effect. When combined, the overall effect would be hypoplasic. There was an example of this epistatic interaction between two QTLs. When BP-lowering alleles at one QTL were experimentally put together with BP-raising alleles at another QTL, the combined effect was the same as for the BP-lowering alleles on Chr 3 (31). This fact indicates that these QTLs do not act alone, but in a hierarchical relationship with each other in controlling the overall BP. The same principle could be applied to the genetic determinants of SMCN.

As to the mechanisms for increasing SMCN, there are two possibilities. The QTL could either favor the SMC proliferation or reduce the SMC death. The present data showing an inverse relationship between DNA fragmentation and SMCN (Table 2) suggest a possible role for reduced apoptosis. Defining the relative contribution of these cellular mechanisms, however, is worthy of further investigations.

Agtr1b is a Candidate Gene for an SMCN QTL
It is noted that Agtr1b is present in the region containing the QTL for SMCN (Fig. 1). Interestingly, we detected two nucleotide differences leading to significant changes in amino acids in congenic strains possessing MNS Agtr1b alleles (i.e., C2S.M, C2S.M1, and C2S.M2), and that possessing the S Agtr1b allele (i.e., C2S.M6; see RESULTS). The presence of these mutations in Agtr1b corresponding to the location of the QTL provides a possible mechanism for increased SMCN, because the pro-growth and anti-apoptotic effects of AT1 receptors on SMC are well known (4).

The mutations found in Agtr1b could have functional roles in controlling SMC proliferation. As an initial investigation of this issue, we chose to measure contractile responses to ANG II, since this is a proximal marker of receptor function. Surprisingly, we observed that responses to ANG II were reduced in C2S.M2 vs. S rats. We speculate that a prior AT1 receptor activation in vivo may have resulted in receptor desensitization in the aortic tissue isolated from C2S.M2 rats. Such a negative effect of in vivo receptor activation on the vascular sensitivity ex vivo seems reminiscent of hypertensive human and rat arteries, where an enhanced local expression of endothelin resulted in a selective attenuation of contractile responses to endothelin in tissues stimulated ex vivo (35). Alternatively, the altered responses to ANG II may be an epiphenomenon. In vitro studies with cultured SMCs are needed to better define the functional correlates of the mutations in Agtr1b and its significance for the SMC proliferation.

Another way to validate whether Agtr1b would remain a good candidate gene or not for the SMCN QTL is by fine congenic mapping. If Agtr1b is the SMCN QTL, no matter how small the region harboring the QTL is minimized to (e.g., 100–200 kb), Agtr1b should always be included in the same region, and this congenic substrain should also have an effect on SMCN. Alternatively, Agtr1b could simply be a marker indicating the approximate location of the QTL for SMCN. The inducible form of the nitric oxide synthase (Nos2) is a case in point. Nos2 was located in a broad region containing a QTL for BP initially, but a fine mapping ruled it out as a QTL because a congenic strain harboring it did not show a BP effect (32). Furthermore, fine congenic mapping will also test the possibility that there are multiple QTLs for SMCN present in the interval between Prlr and D2Rat303.

In summary, a major finding in the present work is that there is a QTL(s) for vascular SMCN that acts independently of hypertension. This discovery has laid the foundation for the identification of the QTL and will lead to revelations of its underlying physiological mechanisms regulating vascular remodeling independently of BP. Agtr1b is a candidate gene worthy of further investigation, using fine mapping and in vitro functional assays, to determine whether it is the SMCN QTL. By use of the same strategy of fine congenic mapping and comparative congenic mapping of BP QTLs, the QTL(s) for SMCN can and will be localized to a smaller interval for candidate cloning (i.e., Agtr1b) or, if not, then positional cloning.


    GRANTS
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by grants from Canadian Institutes for Health Research (CIHR) to A. Y. Deng. A. Y. Deng is an Established Investigator of the American Heart Association, National Center. D. deBlois, J. Tremblay, and P. Hamet are supported by grants from CARDIOGENE and CIHR. D. deBlois is a scholar of the Fonds de la Recherche en Santé du Québec. J. Dutil holds a CIHR graduate fellowship.


    FOOTNOTES
 
Article published online before print. See web site for date of publication (http://physiolgenomics.physiology.org).

Address for reprint requests and other correspondence: A. Deng, Research Centre-Centre Hospitalier de l’Université de Montréal (CHUM), 7-132 Pavillon Jeanne Mance, 3840, rue St. Urbain, Montréal, Québec, H2W 1T8, Canada (e-mail: alan.deng{at}umontreal.ca).

10.1152/physiolgenomics.00063.2004.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Alemayehu A, Breen L, Krenova D, and Printz MP. Reciprocal rat chromosome 2 congenic strains reveal contrasting blood pressure and heart rate QTL. Physiol Genomics 10: 199–210, 2002.[Abstract/Free Full Text]
  2. Ariyarajah A, Palijan A, Dutil J, Prithiviraj K, Deng Y, and Deng AY. Dissecting quantitative trait loci into opposite blood pressure effects on Dahl rat chromosome 8 by congenic strains. J Hypertens 22: 1495–1502, 2004.[CrossRef][ISI][Medline]
  3. Clark JS, Jeffs B, Davidson AO, Lee WK, Anderson NH, Bihoreau MT, Brosnan MJ, Devlin AM, Kelman AW, Lindpaintner K, and Dominiczak AF. Quantitative trait loci in genetically hypertensive rats. Possible sex specificity. Hypertension 28: 898–906, 1996.[Abstract/Free Full Text]
  4. deBlois D, Orlov SN, and Hamet P. Apoptosis in cardiovascular remodeling—effect of medication. Cardiovasc Drugs Ther 15: 539–545, 2001.[CrossRef][ISI][Medline]
  5. deBlois D, Tea BS, Than VD, Tremblay J, and Hamet P. Smooth muscle apoptosis during vascular regression in spontaneously hypertensive rats. Hypertension 29: 340–349, 1997.[Abstract/Free Full Text]
  6. Deng AY. Functional genomics of blood pressure determination: dissecting and assembling a polygenic strait by experimental genetics. Curr Hypertens Rev 1: 35–50, 2005.
  7. Deng AY. In search of hypertension genes in Dahl salt-sensitive rats. J Hypertens 16: 1707–1717, 1998.[CrossRef][ISI][Medline]
  8. Deng AY, Dene H, Pravenec M, and Rapp JP. Genetic mapping of two new blood pressure quantitative trait loci in the rat by genotyping endothelin system genes. J Clin Invest 93: 2701–2709, 1994.[ISI][Medline]
  9. Deng AY, Dene H, and Rapp JP. Mapping of a quantitative trait locus for blood pressure on rat chromosome 2. J Clin Invest 94: 431–436, 1994.[ISI][Medline]
  10. Deng AY, Dene H, and Rapp JP. Congenic strains for the blood pressure quantitative trait locus on rat chromosome 2. Hypertension 30: 199–202, 1997.[Abstract/Free Full Text]
  11. Deng AY, Jackson CM, Hoebee B, and Rapp JP. Mapping of rat chromosome 2 markers generated from chromosome-sorted DNA. Mamm Genome 8: 731–735, 1997.[CrossRef][ISI][Medline]
  12. Deng AY and Rapp JP. Evaluation of the angiotensin II receptor AT1B gene as a candidate gene for blood pressure. J Hypertens 12: 1001–1006, 1994.[ISI][Medline]
  13. Deng Y and Rapp JP. Cosegregation of blood pressure with angiotensin converting enzyme and atrial natriuretic peptide receptor genes using Dahl salt-sensitive rats. Nat Genet 1: 267–272, 1992.[CrossRef][ISI][Medline]
  14. Dominiczak AF, Devlin AM, Lee WK, Anderson NH, Bohr DF, and Reid JL. Vascular smooth muscle polyploidy and cardiac hypertrophy in genetic hypertension. Hypertension 27: 752–759, 1996.[Abstract/Free Full Text]
  15. Dubay C, Vincent M, Samani NJ, Hilbert P, Kaiser MA, Beressi JP, Kotelevtsev Y, Beckmann JS, Soubrier F, Sassard J, and Lathrop GM. Genetic determinants of diastolic and pulse pressure map to different loci in Lyon hypertensive rats. Nat Genet 3: 354–357, 1993.[CrossRef][ISI][Medline]
  16. Dutil J and Deng AY. Further chromosomal mapping of a blood pressure QTL in Dahl rats on chromosome 2 using congenic strains. Physiol Genomics 6: 3–9, 2001.[Abstract/Free Full Text]
  17. Dutil J and Deng AY. Mapping a blood pressure quantitative trait locus to a 5.7-cM region in Dahl salt-sensitive rats. Mamm Genome 12: 362–365, 2001.[CrossRef][ISI][Medline]
  18. Garret MR and Rapp JP. Multiple blood pressure QTL on rat chromosome 2 defined by congenic Dahl rats. Mamm Genome 13: 41–44, 2002.[CrossRef][ISI][Medline]
  19. Garret MR, Zhang X, Dukhanina OI, Deng AY, and Rapp JP. Two linked blood pressure QTL on chromosome 10 defined by Dahl-rat congenic strains. Hypertension 38: 779–785, 2001.[Abstract/Free Full Text]
  20. Guicheney P, Soussan K, Dausse E, and Rota R. Dissociation of hypertension and genetically enhanced cell growth capacity in skin fibroblasts of F2 hybrid spontaneously hypertensive rats/Wistar-Kyoto rats. Am J Hypertens 5: 556–565, 1992.[ISI][Medline]
  21. Hadrava V, Tremblay J, and Hamet P. Abnormalities in growth characteristics of aortic smooth muscle cells in spontaneously hypertensive rats. Hypertension 13: 589–597, 1989.[Abstract]
  22. Hamet P, Kaiser MA, Sun Y, Page V, Vincent M, Kren V, Pravenec M, Kunes J, Tremblay J, and Samani NJ. HSP27 locus cosegregates with left ventricular mass independently of blood pressure. Hypertension 28: 1112–1117, 1996.[ISI][Medline]
  23. Hamet P, Thorin-Trescases N, Moreau P, Dumas P, Tea BS, deBlois D, Kren V, Pravenec M, Kunes J, Sun Y, and Tremblay J. Workshop: excess growth and apoptosis: is hypertension a case of accelerated aging of cardiovascular cells? Hypertension 37: 760–766, 2001.[Abstract/Free Full Text]
  24. Jeffs B, Negrin CD, Graham D, Clark JS, Anderson NH, Gauguier D, and Dominiczak AF. Applicability of a "speed" congenic strategy to dissect blood pressure quantitative trait loci on rat chromosome 2. Hypertension 35: 179–187, 2000.[Abstract/Free Full Text]
  25. Kato N, Hyne G, Bihoreau MT, Gauguier D, Lathrop GM, and Rapp JP. Complete genome searches for quantitative trait loci controlling blood pressure and related traits in four segregating populations derived from Dahl hypertensive rats. Mamm Genome 10: 259–265, 1999.[CrossRef][ISI][Medline]
  26. Lemay J, Hou Y, Tremblay J, and deBlois D. Angiotensin I-converting enzyme activity and vascular sensitivity to angiotensin I in rat injured carotid artery. Eur J Pharmacol 394: 301–309, 2000.[CrossRef][ISI][Medline]
  27. Marchand EL, Der SS, Hamet P, and deBlois D. Caspase-dependent cell death mediates the early phase of aortic hypertrophy regression in losartan-treated spontaneously hypertensive rats. Circ Res 92: 777–784, 2003.[Abstract/Free Full Text]
  28. Masciotra S, Picard S, and Deschepper CF. Cosegregation analysis in genetic crosses suggests a protective role for atrial natriuretic factor against ventricular hypertrophy. Circ Res 84: 1453–1458, 1999.[Abstract/Free Full Text]
  29. Moujahidine M, Dutil J, Hamet P, and Deng AY. Congenic mapping of a blood pressure QTL on chromosome 16 of Dahl rats. Mamm Genome 13: 153–156, 2002.[CrossRef][ISI][Medline]
  30. Owens GK. Differential effects of antihypertensive drug therapy on vascular smooth muscle cell hypertrophy, hyperploidy, and hyperplasia in the spontaneously hypertensive rat. Circ Res 56: 525–536, 1985.[Abstract]
  31. Palijan A, Dutil J, and Deng AY. Quantitative trait loci with opposing blood pressure effects demonstrating epistasis on Dahl rat chromosome 3. Physiol Genomics 15: 1–8, 2003.[Abstract/Free Full Text]
  32. Palijan A, Lambert R, Sivo Z, Dutil J, and Deng AY. Comprehensive congenic coverage revealing multiple BP QTLs on Dahl rat chromosome 10. Hypertension 42: 515–522, 2003.[Abstract/Free Full Text]
  33. Pravenec M, Gauguier D, Schott JJ, Buard J, Kren V, Bila V, Szpirer C, Szpirer J, Wang JM, and Huang H. Mapping of quantitative trait loci for blood pressure and cardiac mass in the rat by genome scanning of recombinant inbred strains. J Clin Invest 96: 1973–1978, 1995.[ISI][Medline]
  34. Samani NJ, Gauguier D, Vincent M, Kaiser MA, Bihoreau MT, Lodwick D, Wallis R, Parent V, Kimber P, Rattray F, Thompson JR, Sassard J, and Lathrop M. Analysis of quantitative trait loci for blood pressure on rat chromosomes 2 and 13. Age-related differences in effect. Hypertension 28: 1118–1122, 1996.[Abstract/Free Full Text]
  35. Schiffrin EL. State-of-the-Art lecture. Role of endothelin-1 in hypertension. Hypertension 34: 876–881, 1999.[Abstract/Free Full Text]
  36. Schork NJ, Krieger JE, Trolliet MR, Franchini KG, Koike G, Krieger EM, Lander ES, Dzau VJ, and Jacob HJ. A biometrical genome search in rats reveals the multigenic basis of blood pressure variation. Genome Res 5: 164–172, 1995.[Abstract]
  37. Sivo Z, Malo B, Dutil J, and Deng AY. Accelerated congenics for mapping two blood pressure quantitative trait loci on chromosome 10 of Dahl rats. J Hypertens 20: 1–9, 2002.[CrossRef][ISI]
  38. Tea BS, Der SS, Touyz RM, Hamet P, and deBlois D. Proapoptotic and growth-inhibitory role of angiotensin II type 2 receptor in vascular smooth muscle cells of spontaneously hypertensive rats in vivo. Hypertension 35: 1069–1073, 2000.[Abstract/Free Full Text]
  39. Tremblay J, Hum DH, Sanchez R, Dumas P, Pravenec M, Krenova D, Kren V, Kunes J, Pausova Z, Gossard F, and Hamet P. TA repeat variation, Npr1 expression, and blood pressure: impact of the Ace locus. Hypertension 41: 16–24, 2003.[Abstract/Free Full Text]
  40. Vincent M, Samani NJ, Gauguier D, Thompson JR, Lathrop GM, and Sassard J. A pharmacogenetic approach to blood pressure in Lyon hypertensive rats. A chromosome 2 locus influences the response to a calcium antagonist. J Clin Invest 100: 2000–2006, 1997.[Abstract/Free Full Text]
  41. Yamori Y, Igawa T, Kanbe T, Kihara M, Nara Y, and Horie R. Mechanisms of structural vascular changes in genetic hypertension: analyses on cultured vascular smooth muscle cells from spontaneously hypertensive rats. Clin Sci (Lond) 61, Suppl 7: 121s–123s, 1981.




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
21/3/362    most recent
00063.2004v1
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Dutil, J.
Articles by Deng, A. Y.
Articles citing this Article
PubMed
PubMed Citation
Articles by Dutil, J.
Articles by Deng, A. Y.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.