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Human essential hypertension is a complex polygenic trait with underlying genetic components that remain unknown. The stroke-prone spontaneously hypertensive rat (SHRSP) is a model of human essential hypertension, and a number of reproducible blood pressure regulation quantitative trait loci have been found to map to rat chromosome 2. The SP.WKYGla2c* congenic strain was produced by introgressing a region of rat chromosome 2 from the normotensive Wistar Kyoto (WKY) strain into the genetic background of the SHRSP. Systolic and diastolic blood pressures were significantly reduced in the SP.WKYGla2c* compared with the SHRSP parental strain (198/134±6.1/3.3 versus 172/120±3.8/3.4 mm Hg; F=15.8/8.1, P =0.0009/0.013). Genome-wide microarray expression profiling was undertaken to identify differentially expressed genes among the parental SHRSP, WKY, and congenic strain. We identified a significant reduction in expression of glutathione S -transferase μ-type 2, a gene involved in the defense against oxidative stress. Quantitative reverse transcription–polymerase chain reaction relative to a β-actin standard confirmed the microarray results with SHRSP mRNA at 8.56×10 −4 ±1.6×10 −4 compared with SP.WKYGla2c* 3.67×10 −3 ±2.8×10 −4 (95% CI −3.9×10 −3 to −1.8×10 −3 ; P =0.0034) and WKY 4.03×10 −3 ±5.1×10 −4 ; (95% CI −5.4×10 −3 to −8.9×10 −4 ; P =0.027). We also identified regions of conserved synteny, each containing the Gstm2 gene, on mouse chromosome 3 and human chromosome 1.

Abstract —The present study was designed to assess vascular smooth muscle cell (VSMC) proliferation and apoptosis in primary cultured VSMCs prepared from the aortic tunica media of adult (4 to 5 months old) age- and gender-matched groups of stroke-prone spontaneously hypertensive rats (SHRSP) and the normotensive reference strain, Wistar-Kyoto (WKY) rats. In the present study, VSMC proliferation was assessed with measurement of DNA synthesis in response to stimulation of G 0 /G 1 arrested VSMCs with 10% serum, whereas apoptosis was measured in response to serum deprivation. Apoptosis in aortic VSMCs was assessed in vitro with the technique of Annexin V binding in combination with propidium iodide exclusion with bivariate flow cytometric analysis. The percentage of necrotic VSMCs in the cell populations was assessed simultaneously. The light-scattering properties of the cells were assessed to provide further information on cell shrinkage and chromatin condensation. Results of the present study have shown enhanced DNA synthesis in VSMCs from SHRSP (n=10; 5.2±0.9 cpm×10 3 /mg protein) compared with WKY (n=12; 2.4±0.7 cpm×10 3 /mg protein; P P

Abstract —Human essential hypertension is a complex, multifactorial, quantitative trait under a polygenic control. Several strategies have been developed over the last decade to dissect genetic determinants of hypertension. Of these, the most successful have been studies that identified rare mendelian syndromes in which a single gene mutation causes high blood pressure. The attempts to identify multiple genes, each with a small contribution to the common polygenic form of hypertension, have been less successful. Several laboratories focused their attention on rat models of genetic hypertension, which can be considered as a reductionist paradigm for human disease. Using numerous crosses between hypertensive and normotensive strains, investigators identified several quantitative trait loci (QTL) for blood pressure subphenotypes and for cardiovascular complications such as left ventricular hypertrophy, kidney failure, stroke, and insulin resistance. Furthermore, congenic strains have been produced to confirm the existence of some of these QTL and to narrow down the chromosomal regions of interest. A number of interesting strategies have been developed, including a “speed” congenic strategy perfected by our group in Glasgow. However, the limit of congenic strategy is estimated at 1 cM, which corresponds to 2×10 6 base pairs of DNA and ≈50 candidate genes. It is envisaged that gene expression profiling with cDNA microarrays might allow a quick progression toward the gene identification within cardiovascular QTL. In parallel experimental effort, several laboratories have been developing gene transfer/therapy strategies with adenoviral or adeno-associated viral vectors used, for example, to overexpress protective vascular genes such as vascular endothelial growth factor or endothelial nitric oxide synthase. It is anticipated that further developments in positional cloning of susceptibility and severity genes in hypertension and its complications will lead to a direct transfer of these discoveries to essential hypertension in humans and will ultimately produce novel targets for local and systemic gene therapy in cardiovascular disease.

Abstract —The identification of any quantitative trait locus (QTL) via a genome scan is only the first step toward the ultimate goal of gene identification. The next step is the production of congenic strains by which the existence of a QTL may be verified and the implicated chromosomal region be reduced to a size applicable to positional cloning of the causal gene. We used a speed congenic breeding protocol previously verified in mice for 2 blood pressure QTLs on rat chromosome 2. Four congenic strains were produced through introgression of various segments of chromosome 2 from Wistar-Kyoto rats from Glasgow colonies [WKY (Gla) rats] into the recipient stroke-prone spontaneously hypertensive rats from Glasgow colonies [SHRSP (Gla) ], and vice versa. The number of backcross generations required for each strain to achieve complete homozygosity at 83 background genetic markers in a “best” male varied between 3 and 4. Transfer of the region of rat chromosome 2 containing both QTLs from WKY (Gla) into an SHRSP (Gla) genetic background lowered both baseline and salt-loaded systolic blood pressure by ≈20 and ≈40 mm Hg in male congenic rats compared with the SHRSP parental strain ( F =53.4, P F =28.0, P < 0.0005, respectively). In contrast, control animals for stowaway heterozygosity presented no deviation from the blood pressure values recorded for the SHRSP (Gla) , indicating that if such heterozygosity exists, its effect on blood pressure is negligible. A reciprocal strategy in which 1 or both QTLs on rat chromosome 2 were transferred from SHRSP (Gla) into a WKY (Gla) genetic background resulted in statistically significant but smaller blood pressure increases for 1 of these QTLs. These results confirm the existence of blood pressure QTLs on rat chromosome 2 and demonstrate the applicability of a speed congenic strategy in the rat and emphasize the important role of the genetic background.

Abstract —We have investigated genetic transmission of increased sensitivity to focal cerebral ischemia and the influence of gender in the stroke-prone spontaneously hypertensive rat (SHRSP). Halothane-anesthetized, 3- to 5-month-old male and female Wistar-Kyoto rats (WKY), SHRSP, and the first filial generation rats (F 1 crosses 1 and 2) underwent distal (2 mm) permanent middle cerebral artery occlusion (MCAO) by electrocoagulation. Infarct volume was measured by using hematoxylin-eosin–stained sections and image analysis 24 hours after ischemia and expressed as a percentage of the volume of the ipsilateral hemisphere. Infarct volume in males and females grouped together were significantly larger in SHRSP, F 1 cross 1 (SHRSP father), and F 1 cross 2 (WKY father), at 36.6±2.3% (mean±SEM, P P P 1 cross 1 (18.9±2.4%, n=6) developed significantly smaller infarcts than male F 1 cross 2 (32.8±2%, n=8, P 1 cross 1 and cross 2 hybrids, suggesting a dominant or codominant trait; response to cerebral ischemia appears to be affected by gender and stage in the estrous cycle. In addition, the male progenitor of the cross (ie, SHRSP versus WKY) influences stroke sensitivity in male F 1 cohorts.

Abstract —Previous studies suggested that atrial natriuretic peptide gene ( Anp) and brain natriuretic peptide gene ( Bnp) are plausible candidate genes for susceptibility to stroke and for sensitivity to brain ischemia in the stroke-prone spontaneously hypertensive rat (SHRSP). We performed structural and functional analyses of these 2 genes in SHRSP from Glasgow colonies (SHRSP Gla ) and Wistar-Kyoto rats from Glasgow colonies (WKY Gla ) and developed a radiation hybrid map of the relevant region of rat chromosome 5. Sequencing of the coding regions of the Anp and Bnp genes revealed no difference between the 2 strains. Expression studies in brain tissue showed no differences at baseline and at 24 hours after middle cerebral artery occlusion. Plasma concentrations of atrial natriuretic peptide (ANP) did not differ between the SHRSP Gla and WKY Gla , whereas concentrations of brain natriuretic peptide were significantly higher in the SHRSP Gla as compared with the WKY Gla (n=11 to 14; 163±21 pg/mL and 78±14 pg/mL; 95% confidence interval 31 to 138, P =0.003). We did not detect any attenuation of endothelium-dependent relaxations to bradykinin or ANP in middle cerebral arteries from the SHRSP Gla ; indeed the sensitivity to ANP was significantly increased in arteries harvested from this strain (WKY Gla : n=8; pD 2 =7.3±0.2 and SHRSP Gla : n=8; pD 2 =8.2±0.15; P Anf marker in the telomeric position of rat chromosome 5 in close proximity to D5Rat48, D5Rat47, D5Mgh15, and D5Mgh16. These results exclude Anp and Bnp as candidate genes for the sensitivity to brain ischemia and pave the way to further congenic and physical mapping strategies.

54 Background Localisation of QTL using a genome wide scan strategy is the first step towards gene identification. This is followed by the construction of congenics by which the existence of the QTL can be verified. Congenic strains for chromosome 2 using the Dahl S as the recipient and WKY and MNS as donors, showed variable blood pressure (BP) depending on the donor alleles 1 . Methods We used a speed congenic approach to produce several congenic strains of chromosome 2 using the SHRSP Gla and the WKY Gla as recipients to test the effect of the genetic background of a given congenic on BP. In some strains, the region introgressed was identical to avoid confounding effects of additional QTLs. Two reciprocal control congenic strains were also produced to determine the presence of any passenger loci. Baseline systolic BP was measured by radiotelemetry. Results Four congenic and 2 parental strains were phenotyped (n=3-6). Transfer of the region of rat chromosome 2 from the WKY into a SHRSP background significantly lowered both day and night-time systolic BP by approximately 16 and 20 mm Hg respectively in male congenic rats compared to the SHRSP parental strains (pet al . Hypertension 1997;30:199-202