George T. Cicila

Sequence Variation of Bovine Elastin mRNA Due to Alternative Splicing

Poly A+ RNA, isolated from a single 210 day fetal bovine nuchal ligament, was used to synthesize cDNA by the RNase H method, using AMV reverse transcriptase for first strand synthesis and DNA polymerase I for the second strand. The cDNA was inserted into lambda gt10 using EcoRI linkers, and recombinant phage containing elastin sequences were identified by hybridization with a 1.3 kb sheep elastin cDNA clone, pcSELI (Yoon, K. et al., Biochem. Biophys. Res. Comm. 118: 261-265, 1984). Three clones containing the largest inserts of 2.9, 2.8, and 2.6 kb were selected for further study. The complete sequence analysis of the 3 clones was correlated with the sequence of 10.2 kb of the bovine elastin gene. The analyses: (i) showed that the cDNA encompassed the great majority of the translated sequence, (ii) ordered the tryptic peptides of porcine tropoelastin, (iii) determined new amino acid sequences not previously found in the porcine peptides and (iv) demonstrated that alternative splicing of the primary transcript leads to significant variation in the sequence of the translated portion of the mRNA.

Blood pressure and survival of a Chromosome 7 congenic strain bred from Dahl rats

Abstract11 β-hydroxylase (Cyp11b1) mutations were previously linked to altered steroid biosynthesis and blood pressure in Dahl salt-resistant (R) and Dahl salt-sensitive (S) rats. In the present work, interval mapping identified a putative blood pressure quantitative trait locus (QTL) near Cyp11b1 in an F1(SxR)xS population (LOD = 2.0). Congenic rats (designated S.R-Cyp11b) were constructed by introgressing the R-rat Cyp11b1 allele into the S strain. S.R-Cyp11b rats had significantly lower blood pressure and heart weight compared with S rats, proving the existence of a blood pressure QTL on Chromosome (Chr) 7 despite the fact that QTL linkage analysis of blood pressure never achieved stringent statistical criteria for significance. To test the effects of the introgressed region on blood pressure and survival, S.R.-Cyp11b and S rats were maintained on a 4% NaC1 diet until they died or became moribund. Analysis of variance (ANOVA) indicated significant strain differences in blood pressure and days survived (P < 0.0001 for both) as well as gender differences in days survived (P = 0.0003). Kaplan-Meier survival analysis also found significant strain (P < 0.0001) and gender (P = 0.007) differences in days survived. However, when the effects of blood pressure were removed, significant strain differences in survival essentially disappeared. This suggests that the increased survival of S.R-Cyp11b rats was largely due to their decreased blood pressure and thus strongly corroborates the existence of a blood pressure QTL on Chr 7 near or at Cyp11b1.

VALVULAR INTERSTITIAL CELLS EXPRESS ANGIOTENSINOGEN AND CATHEPSIN D, AND GENERATE ANGIOTENSIN PEPTIDES

Cells capable of de novo angiotensin (Ang)II generation in the heart remain unidentified. High-density angiotensin converting enzyme (ACE) binding has been localized to sites of high collagen turnover, such as heart valve leaflets and their valvular interstitial cells (VIC). VIC express ACE mRNA and their membrane-bound ACE utilizes AngI as substrate. Whether VIC also express angiotensinogen (Ao) and an aspartyl protease, and whether they generate AngI and II de novo, is presently unknown. We sought to address these questions in serum-deprived cultured VIC. Ao, renin and cathepsin D (Cat-D) mRNA expression was addressed by RT-PCR. Production of Ao, AngI and AngII peptides were measured in VIC-culture media by radioimmunoassay (RIA). Immunoreactive Cat-D was detected by immunofluorescein labeling and Western blotting. Cat-D and renin activities were determined by spectrofluorometric and autoradiographic methods and AngI generation by RIA. Results showed (a) expression of Ao and Cat-D both at mRNA and protein levels; (b) AngI and AngII peptides in culture media; (c) acceleration of AngII production by exogenous AngI (1 nmol/l), which was blocked by lisinopril (0.1 mumol/l); (d) that dexamethasone (0.1 mumol/l) increased AngII production; (e) a 46 kDa immunoreactive Cat-D protein by Western blotting; (f) aspartyl protease activity, using chromogenic and 125I-labeled Ao as substrates, inhibited by pepstatin-A; and (g) the absence of renin mRNA and activity. It is concluded that at both the mRNA and protein levels, cultured VIC express Ao and Cat-D, and can generate AngI and AngII peptides by the action of a non-renin protease Cat-D and ACE, respectively. VIC therefore appear to represent a constitutive nonendothelial cell found in adult rat heart valve leaflets, which are capable of de novo Ang peptide generation.

Substitution Mapping in Dahl Rats Identifies Two Distinct Blood Pressure Quantitative Trait Loci Within 1.12- and 1.25-Mb Intervals on Chromosome 3

Substitution mapping was used to refine the localization of blood pressure (BP) quantitative trait loci (QTL) within the congenic region of S.R-Edn3 rats located at the q terminus of rat chromosome 3 (RNO3). An F2(S × S.R-Edn3) population (n = 173) was screened to identify rats having crossovers within the congenic region of RNO3 and six congenic substrains were developed that carry shorter segments of R-rat-derived RNO3. Five of the six congenic substrains had significantly lower BP compared to the parental S rat. The lack of BP lowering effect demonstrated by the S.R(ET3 × 5) substrain and the BP lowering effect retained by the S.R(ET3 × 2) substrain together define the RNO3 BP QTL-containing region as ∼4.64 Mb. Two nonoverlapping substrains, S.R(ET3 × 1) and S.R(ET3 × 6), had significantly lower BP compared to the S strain, indicating the presence of two distinct BP QTL in the RNO3 q terminus. The RNO3 q terminus was fine mapped with newly developed polymorphic markers to characterize the extent of the congenic regions. The two RNO3 BP QTL regions were thus defined as within intervals of 0.05–1.12 and 0.72–1.25 Mb, respectively. Also important was our difficulty in fine mapping and marker placement in this portion of the rat genome (and thus candidate gene identification) using the available genomic data, including the rat genome sequence.

Epistatic genetic determinants of blood pressure and mortality in a salt-sensitive hypertension model.

Although genetic determinants protecting against the development of elevated blood pressure (BP) are well investigated, less is known regarding their impact on longevity. We concomitantly assessed genomic regions of rat chromosomes 3 and 7 (RNO3 and RNO7) carrying genetic determinants of BP without known epistasis, for their independent and combinatorial effects on BP and the presence of genetic determinants of survival using Dahl salt-sensitive (S) strains carrying congenic segments from Dahl salt-resistant (R) rats. Although congenic and bicongenic S.R strains carried independent BP quantitative trait loci within the RNO3 and RNO7 congenic regions, only the RNO3 allele(s) independently affected survival. The bicongenic S.R strain showed epistasis between R-rat RNO3 and RNO7 alleles for BP under salt-loading conditions, with less-than-additive effects observed on a 2% NaCl diet and greater-than-additive effects observed after prolonged feeding on a 4% NaCl diet. These RNO3 and RNO7 congenic region alleles had more-than-additive effects on survival. Increased survival of bicongenic compared with RNO3 congenic rats was attributable, in part, to maintaining lower BP despite chronic exposure to an increased dietary salt (4% NaCl) intake, with both strains showing delays in reaching highest BP. R-rat RNO3 alleles were also associated with superior systolic function, with the S.R bicongenic strain showing epistasis between R-rat RNO3 and RNO7 alleles leading to compensatory hypertrophy. Whether these alleles affect survival by additional actions within other BP-regulating tissues/organs remains unexplored. This is the first report of simultaneous detection of independent and epistatic loci dictating, in part, longevity in a hypertensive rat strain.

Blood pressure and proteinuria effects of multiple quantitative trait loci on rat chromosome 9 that differentiate the spontaneously hypertensive rat from the Dahl salt-sensitive rat

A blood pressure (BP) quantitative trait locus (QTL) was previously located within 117 kb on rat chromosome 9 (RNO9) using hypertensive Dahl salt-sensitive and normotensive Dahl salt-resistant rats. An independent study between two hypertensive rat strains, the Dahl salt-sensitive rat and the spontaneously hypertensive rat (SHR), also detected a QTL encompassing this 117 kb region. Dahl salt-sensitive alleles in both of these studies were associated with increased BP. To map SHR alleles that decrease BP in the Dahl salt-sensitive rat, a panel of eight congenic strains introgressing SHR alleles onto the Dahl salt-sensitive genetic background were constructed and characterized. S.SHR(9)x3B, S.SHR(9)x3A and S.SHR(9)x2B, the congenic regions of which span a portion or all of the 1 logarithm of odds (LOD) interval identified by linkage analysis, did not significantly alter BP. However, S.SHR(9), S.SHR(9)x4A, S.SHR(9)x7A, S.SHR(9)x8A and S.SHR(9)x10A, the introgressed segments of which extend distal to the 1 LOD interval, significantly reduced BP. The shortest genomic segment, BP QTL1, to which this BP-lowering effect can be traced is the differential segment of S.SHR(9)x4A and S.SHR(9)x2B, to which an urinary protein excretion QTL also maps. However, the introgressed segment of S.SHR(9)x10A, located outside of this QTL1 region, represented a second BP QTL (BP QTL2) having no detectable effects on urinary protein excretion. In summary, the data suggest that there are multiple RNO9 alleles of the SHR that lower BP of the Dahl salt-sensitive rat with or without detectable effects on urinary protein excretion and that only one of these BP QTLs, QTL1, overlaps with the 117 kb BP QTL region identified using Dahl salt-sensitive and Dahl salt-resistant rats.

Regulation of the genes for 11β-hydroxysteroid dehydrogenase type 1 and type 2 in the kidney of the Dahl rat

BACKGROUND An isoenzyme of 11beta-hydroxysteroid dehydrogenase (11beta-HSD), 11beta-HSD-2 confers aldosterone specificity on the mineralocorticoid receptor (MR) and is found collocated in renal cortical collecting duct cells with the MR. To investigate whether the salt sensitivity of the Dahl salt-sensitive (S) rat is due to 11beta-HSD deficiency, we measured 11beta-HSD-1 and 11beta-HSD-2 mRNA levels in the kidneys of Dahl-S and Dahl salt-resistant (R) rats. In addition, we studied the effects of gender, age and dietary sodium on expression of mRNA for the two isoforms. S and R rats were placed on low- or high-sodium (HNa) diets and sacrificed after 33 and 115 days. Rat kidney RNA was isolated and 11beta-HSD-1 and 11beta-HSD-2 mRNA levels were measured on Northern filter hybridization using isoform-specific probes. RESULTS No strain differences were observed in the mRNA expression of the two isoforms of 11beta-HSD under any of the experimental conditions. No gender or age differences were observed in 11beta-HSD-2 mRNA but HNa diet almost doubled 11beta-HSD-2 mRNA (P<0.0009). 11beta-HSD-1 mRNA levels were consistently higher, more than double, in male rats versus females rats (P<0.0001), and in the 115-day-old rats versus the 33-day-old rats (P<0.0001). Dietary sodium intake did not affect 11beta-HSD-1 mRNA levels. CONCLUSIONS There is no difference in the expression of the two isoforms of 11beta-HSD in the kidneys of the S and R rats, which might explain the salt sensitivity and higher blood pressure of the S rat. Renal 11beta-HSD-1 mRNA levels are higher in male than in female rats, and in the older rats of both strains. In the kidney, the 11beta-HSD-2 gene is regulated by sodium status but is not affected by gender or age.

Endothelin A and B Receptors Are Down-Regulated in the Hearts of Hypertensive Rats

Endothelins are vasoactive pep-tides that have been implicated in the development and maintenance of systemic arterial hypertension. The biologic effects of endothelins result from activation of either or both of the two known endothelin receptor subtypes, A and B [ET-R(A) and ET-R(B)], which are present not only in blood vessels but also throughout the cardiovascular and central nervous systems. To investigate the potential role and regulation of myocardial endothelin receptors in hypertension, we examined the expression of ET-R(A) and ET-R(B) receptors in the hearts of normotensive and hypertensive rats. A cDNA probe for the ET-R(A) receptor was obtained by polymerase chain reaction amplification of rat aortic smooth muscle cell mRNA, using degenerate primers specific for intramembrane domains III and VI of G-coupled receptors. Moderate stringency hybridization screening of a rat aortic smooth muscle cell cDNA library yielded a partial clone for the ET-R(B) receptor. These two clones wereusedto examine expression of the ET-R(A) and ET-R(B) receptors in heart, brain, and kidney tissues from Wistar-Kyoto (normotensive), spontaneously hypertensive, salt-hypertensive sensitive, and salt-hypertensive resistant rats by Northern analysis. ET-R(A) and ET-R(B) mRNA were present in the hearts of normal rats. Spontaneously hypertensive rat hearts did not express either ET-R(A) or ET-R(B) mRNA, whereas both salt-hypertensive sensitive and resistant rats fed a high-salt diet expressed both ET-R(A) and ET-R(B) receptor mRNAs. Conversely, in the brain of spontaneously hypertensive rats, mRNAs for both ET-R(A) and ET-R(B) mRNA were present. These observations argue for tissue-specific expression of ET-R(A) and ET-R(B) receptors in spontaneously hypertensive rats, and for differences in endothelin receptor regulation among different models of hypertension in the rat.

Neointimal Hyperplasia and Vasoreactivity Are Controlled by Genetic Elements on Rat Chromosome 3

Neointimal hyperplasia (NIH) can lead to restenosis after clinical vascular interventions. NIH results from complex and poorly understood interactions between signaling cascades in the extracellular matrix and the disrupted endothelium, which lead to vessel occlusion. Quantitative trait loci (QTLs) were reported previously on rat chromosomes 3 and 6 through linkage analysis of postinjury NIH in midiliac arterial sections. In the current study, substitution mapping validated the RNO3 NIH QTL but not the RNO6 NIH QTL. The SHR.BN3 congenic strain had a 3-fold increase in the percentage of NIH compared with the parental spontaneously hypertensive rat strain. A double congenic study of RNO3+RNO6 NIH QTL segments suggested less than additive effects of these 2 genomic regions. To test the hypothesis that changes in vessel dynamics account for the differences in NIH formation, we performed vascular reactivity studies in the Brown Norway (BN), spontaneously hypertensive rat (SHR), SHR.BN3, and SHR.BN6 strains. De-endothelialized left common carotid artery rings of the SHR.BN3 showed an increased vascular responsiveness when treated with serotonin or prostaglandin F2α, with significant differences in EC50 and maximum effect (P<0.01) values compared with the spontaneously hypertensive rat parental strain. Because both vascular reactivity and percentage of NIH formation in the SHR.BN3 strain are significantly higher than the SHR strain, we postulate that these traits may be associated and are controlled by genetic elements on RNO3. In summary, these results confirm that the RNO3 NIH QTL carries the gene(s) contributing to postinjury NIH formation.

An improved linkage map of rat Chromosome 3 with three mapping panels

Abstract. Our purposes were to develop an improved linkage map for rat Chromosome 3 and to develop new markers polymorphic between Dahl salt-sensitive (S) and Dahl salt-resistant (R) rats. The linkage mapping panel consisted of three F2 populations totaling 359 rats. Twenty-five new markers were developed and placed on the linkage map. About half of these markers (13) were polymorphic between S and R rats. The final map spans 124.7 centiMorgans (cM) and includes 64 markers. The average distance between adjacent markers is 1.9 cM, and the largest separation is 10.5 cM.