Chromosomal mapping of quantitative trait loci contributing to stroke in a rat model of complex human disease.

Stroke is a complex disorder with a poorly understood multifactorial and polygenic aetiology. We used the stroke-prone spontaneously hypertensive rat (SHRSP) as a model organism, mated it with the stroke-resistant spontaneously hypertensive rat (SHR) and performed a genome-wide screen in the resultant F2 cohort where latency until stroke, but not hypertension (a major confounder) segregated. We identified three major quantitative trait loci, STR1-3, with lod scores of 7.4, 4.7 and 3.0, respectively, that account for 28% of the overall phenotypic variance. STR2 colocalizes with the genes encoding atrial and brain natriuretic factor, peptides with important vasoactive properties. Our results demonstrate the existence of primary, blood pressure-independent genetic factors predisposing to a complex form of stroke.

Association and cosegregation of stroke with impaired endothelium-dependent vasorelaxation in stroke prone, spontaneously hypertensive rats.

While hypertension is a major risk factor for stroke, it is not its sole determinant. Despite similar blood pressures, spontaneously hypertensive rats (SHR) do not share the predisposition to cerebrovascular disease typical of stroke-prone spontaneously hypertensive rats (SHRSP). We investigated vascular function in male SHR and SHRSP as well as in SHRSP/SHR-F2 hybrid animals. Animals were maintained on the appropriate dietary regimen necessary for the manifestation of stroke. Among the hybrid animals, a group of stroke-prone and a group of stroke-resistant rats were selected. Blood pressure was similar in all groups. Endothelium-independent vascular reactivity tested on isolated rings of thoracic aorta and basilar artery after death showed similar contractile and dilatory responses to serotonin and nitroglycerin, respectively, in all groups. In contrast, endothelium-dependent relaxation, in response to acetylcholine or substance P, was markedly reduced in SHRSP compared with SHR. Similarly, reduced vasodilatory responses were present in aortae of F2 rats that had suffered a stroke when compared with SHR or F2 rats resistant to stroke. The observed association and cosegregation of stroke with significant and specific impairment of endothelium-dependent vasorelaxation among SHRSP and stroke-prone F2 hybrids, respectively, suggest a potential causal role of altered endothelium-dependent vascular relaxation in the pathogenesis of stroke.

Contribution of the sympathetic nervous system to the hypertensive effect of a high sodium diet in stroke-prone spontaneously hypertensive rats.

In stroke-prone spontaneously hypertensive rats (SHRSP) plasma norepinephrine levels and vascular reactivity to norepinephrine are increased and intravascular volume is reduced during the developmental phase of hypertension. Since the accelerated rise in blood pressure following sodium-loading in SHRSP cannot be attributed to the volume-retaining properties of sodium, the effects of an increased dietary intake of sodium on biochemical parameters of sympathetic vascular tone were investigated. The following results were obtained. First, the increased reactivity of vascular smooth muscle was further augmented in sodium-treated SHRSP; the degree of supersensitivity was positively correlated to the plasma sodium concentration. After blockade of the neuronal uptake by 30 microM cocaine, no difference in vascular reactivity to norepinephrine was detected between SHRSP on a normal and SHRSP on a high-sodium diet. Second, the inactivation of norepinephrine by the neuronal uptake was impaired in rats on a high-sodium diet, the impairment being more pronounced in SHRSP than in Wistar-Kyoto (WKY) rats. This decreased inactivation could be expected to cause higher concentrations of the neurotransmitter at the receptor site if the transmitter release from the nerve ending remains constant. Third, the release of norepinephrine and epinephrine into the plasma was increased in sodium-loaded SHRSP but not in sodium-loaded WKY. Cold exposure exaggerates these differences between normotensive and hypertensive rats. These findings suggest that a high-sodium intake modifies the transmission of sympathetic impulses at the level of the nerve terminal in both WKY and SHRSP. In the normotensive rats, moderate impairment of norepinephrine inactivation, however, was balanced by an appropriate reduction in central sympathetic discharge following sodium-loading. In the hypertensive rats, the peripheral disturbance in norepinephrine inactivation due to sodium-loading was obviously not balanced by an adequate withdrawal of central sympathetic discharge. The resultant hemodynamic change was a further increase in the sympathetically mediated vasoconstriction, which is regarded as at least one of the main mechanisms of the sodium-dependent acceleration of hypertension in SHRSP.

Development of a new strain of spontaneously hypertensive rats homozygous for hypothalamic diabetes insipidus.

The aim of the present study was to investigate whether the presence of arginine vasopressin (AVP) is necessary for the establishment of high blood pressure in spontaneously hypertensive rats (SHR). For this purpose we crossbred SHR of the stroke-prone substrain (SHRSP) with rats homozygous for hypothalamic diabetes insipidus of the Brattleboro strain (DI) which are unable to synthetize AVP. The successful introduction of the DI gene into the SHRSP strain (SHRDI) was demonstrated by the following observations: In 10-month-old rats, water intake was similarly elevated in SHRDI as in DI rats (137 +/- 6.5 vs 125 +/- 10.5 ml per 24 hours). AVP was undetectable in the plasma, in the hypothalamus, and in the pituitary of SHRDI and DI rats. Urine osmolality and urinary concentration of sodium and potassium were markedly reduced. SHRDI and DI did not adequately concentrate their urine during an 8-hour period of water deprivation, but both strains of rats responded well with a fall in urine output and a rise in urine osmolality to subcutaneous administration of the non-pressor analog of AVP, DDAVP. Mean arterial blood pressure was markedly increased in SHRDI as well as in SHRSP (184 +/- 9.7 vs 197 +/- 5.2 mm Hg). Thus, we have developed a new line of spontaneously hypertensive rats homozygous for hypothalamic diabetes insipidus. From this finding it is concluded that AVP is not essential for the development and maintenance of spontaneous hypertension of rats.

Hemodynamics and metabolism in stroke-prone spontaneously hypertensive rats before manifestation of brain infarcts.

Genomic screening of hybrids from stroke-prone (SHR-SP) and stroke-resistant spontaneously hypertensive rats (SHR) identified a STR1 locus on the rat chromosome 1, which correlates with the susceptibility to cerebral stroke but not with hypertension. The authors examined whether this genetic abnormality is associated with hemodynamic or metabolic alterations in the brain that can be detected before the manifestation of brain infarction. Starting at 6 weeks of age, SHR-SP were fed with a salt-rich diet to accelerate arterial hypertension. At the age of 12 weeks, animals developed functional symptoms and were age-matched with symptom-negative SHR-SP to differentiate between presymptomatic and postsymptomatic changes. Brains were investigated by multiparametric imaging comprising quantitative double-tracer autoradiography of CBF and cerebral protein synthesis (CPS); bioluminescence imaging of regional ATP, glucose, and lactate content; and umbelliferone fluoroscopic imaging of tissue pH. None of the animals exhibited focal hemodynamic or biochemical abnormalities. In symptom-negative SHR-SP, global CBF was 1.1+/-0.3 mL x g(-1) x min(-1), cortical CPS was 10.1+/-3.1 nmol x g(-1) x min(-1), and cortical ATP, glucose, lactate, and pH levels were in the normal range. In SHR-SP with functional symptoms, ATP, glucose, and lactate levels also were normal, but tissue pH exhibited periventricular alkalosis, CBF was significantly reduced to 0.7+/-0.2 mL x g(-1) x min(-1) (P < 0.001), and cortical CPS was significantly reduced to 6.7+/-2.1 nmol x g(-1) x min(-1) (P < 0.001). The decline in brain perfusion of SHR-SP correlated significantly with both the severity of functional deficits and the decline of protein synthesis. Our observations demonstrate that SHR-SP had already developed functional symptoms before the manifestation of overt brain infarcts and that the symptoms are initiated by a decline in global CBF and cortical CPS. Genetic abnormalities in SHR-SP are associated with a diffuse vascular process that results in global decompensation of blood flow well before the onset of focal brain infarction.

Iso-renin of extrarenal origin. "The tissue angiotensinogenase systems".

Enzymes, similar to kidney renin, are present in extrarenal tissue of most mammals; they hydrolyze angiotensinogen to form angiotensin I. We suggest that these enzymes be called angiotensinogenases. Angiotensinogenase concentrations in extrarenal tissue can exceed those in the kidney. The enzyme has been obtained in pure crystalline form. Angiotensinogenases are part of a complex enzyme system which leads to local production of angiotensin. Results indicating a biologic role of the angiotensinogenase system in brain, adrenal gland, uterus and tissue culture are discussed.

Differential brain atrial natriuretic peptide expression co-segregates with occurrence of early stroke in the stroke-prone phenotype of the spontaneously hypertensive rat.

OBJECTIVE: To determine how the downregulation of atrial natriuretic peptide (ANP) gene expression, previously demonstrated to occur only in the brain of the stroke-prone spontaneously hypertensive rat (SHRsp), in contrast to the stroke-resistant SHR (SHRsr), co-segregates with stroke occurrence in SHRsp/SHRsr F2 descendants in order to study the 'protective' role towards stroke previously demonstrated in SHRsp for the quantitative trait locus STR2 that also carries the ANP gene. DESIGN AND METHODS: Eight male SHRsp, eight male SHRsr and 16 male SHRsp/SHRsr F2-intercross animals (progeny of brother/sister mated F1 hybrids from an original cross between F0 SHRsp and SHRsr) were selected for this study. All rats were exposed to a stroke-permissive Japanese-style diet starting at the age of 6 weeks. Half of the F2 animals had early strokes; the remainder had late strokes. Blood pressure was measured before sacrifice. Analysis of brain ANP expression using an RNase protection assay was performed in all animals. RESULTS: Downregulation of brain ANP in the stroke-prone phenotype was found to co-segregate with the occurrence of early strokes in the F2 rats independently of blood pressure levels. CONCLUSIONS: The observed lower expression of ANP in the brains of stroke-prone rats appears to be the result of an inhibitory effect by another gene or genes. It seems unlikely that this specific trait represents a primary protective mechanism.

Haemodynamic effects of intact digoxin antibody and its Fab fragments in experimental hypertension.

The effects of i.v. injection of intact digoxin antibody (0.3 mg/rat) and of its Fab fragment (40 mg/rat) on blood pressure, cardiac output and total peripheral resistance were measured in conscious spontaneously hypertensive and deoxycorticosterone hypertensive rats. In vitro findings showed that Fab fragment bound radio-labelled digoxin, digitoxin and ouabain more efficiently than did intact antibody. In vivo, Fab fragment prevented the increase of total peripheral resistance induced by i.v. injection digoxin. However, Fab fragment of digoxin antibody did not alter blood pressure, cardiac output or total peripheral resistance in normal and salt-loaded spontaneously hypertensive rats (SHR) in uraemic SHR and in deoxycorticosterone hypertensive rats. We confirmed that intact digoxin antibody--bearing Fc domains with complement activating properties--lowered blood pressure in SHR and in deoxycorticosterone hypertension. This was due to a decrease in total peripheral resistance. Our data suggest that a circulating endogenous digitalis-like factor is unlikely to be important in blood pressure regulation in salt-loaded hypertension in the rat.

Sexual dimorphism of blood pressure in spontaneously hypertensive rats: effects of anti-androgen treatment.

The mechanisms resulting in the greater predisposition of male subjects towards hypertension were investigated in different strains of rats with genetic hypertension [spontaneously hypertensive rats of the stroke-prone strain (SHRSP) and spontaneously hypertensive rats (SHR)] and their respective normotensive controls. Blood pressure was reduced in young (9 weeks of age) hypertensive rats by (1) surgical castration, (2) treatment with the testosterone receptor antagonist cyproterone acetate (CPA), which does not elevate testosterone, or (3) with the testosterone receptor antagonist flutamide, which leads to a feedback elevation of gonadotrophic hormones and plasma testosterone. These treatments had no effect on high blood pressure in old hypertensive rats aged 25 weeks. Both androgen receptor antagonists attenuated high blood pressure development when given for the first 10 days after birth. These data clearly relate the sexual dimorphism of hypertension to testosterone produced during male brain maturation in the early phase of hypertension development. Testosterone appears not to contribute directly to the maintenance of high blood pressure in established hypertension.

Analysis of the genetic basis of the endothelium-dependent impaired vasorelaxation in the stroke-prone spontaneously hypertensive rat: a candidate gene approach.

OBJECTIVE: To investigate the role of potential candidate genes in the pathogenesis of the endothelium-dependent impaired vasorelaxation that associates and co-segregates with stroke in the stroke-prone spontaneously hypertensive rat (SHRsp) compared with the stroke-resistant SHR (SHRsr). DESIGN AND METHODS: An SHRsp/SHRsr F2-intercross (n = 137; 64 males, 73 females) was obtained and, at the age of 6 weeks, it was placed under a stroke permissive Japanese-style diet for 4 weeks. At the end of the treatment the vascular function of each rat was characterized. The maximal vasorelaxation to acetylcholine after maximal vasoconstriction (delta ratio) was considered as the quantitative phenotype. The following candidate genes were related to the delta ratio: renin, angiotensinogen, angiotensin-converting enzyme, angiotensin II AT1b receptor, atrial natriuretic peptide, brain natriuretic peptide, atrial natriuretic peptide GC-A receptor, kallikrein, endothelial nitric oxide synthase. In addition, polymorphic markers located inside areas of the rat genome where other candidates (i.e. adrenomedullin, endothelin, Ang II AT1a receptor) are known to map were included. RESULTS: The endothelial vascular dysfunction of the SHRsp showed a variable distribution among SHRsp/SHRsr F2 descendants, independently from the blood pressure levels. A genotype/phenotype co-segregation analysis for each of the genes tested did not show any statistically significant co-segregation with the vascular phenotype. CONCLUSION: A candidate gene approach used to investigate the genetic basis of the endothelial-dependent vascular dysfunction of the SHRsp strain did not reveal any evidence to support the hypothesis that the genes tested play any role in the pathogenesis of the stroke-related vascular abnormality.

Blood pressure, electrolyte and adrenal responses in swim-trained hypertensive rats.

In order to test the effect of aerobic training on blood pressure, and to examine the putative mechanisms involved, stroke-prone spontaneously hypertensive rats (SHR-SP), borderline hypertensive rats (BHR), and Wistar-Kyoto control rats (WKY) were swim-trained for up to 1.5 h twice-daily for 22 weeks. The BHR were F1 back-cross SHR-SP, WKY. A training effect was observed in the trained rats compared to controls, as demonstrated by slower heart rates, heavier hearts and increased cytochrome oxidase activity in their skeletal muscle. Trained SHR-SP and BHR had significantly lower blood pressures at the end of the intervention period (approximately 10 mmHg) compared to controls. Acute increases in blood pressure with swimming were less in trained than in untrained rats. Trained rats had higher extracellular sodium values than untrained rats. Further, trained SHR-SP and BHR had lower intra-erythrocyte sodium values than controls. Increases in corticosterone, epinephrine and norepinephrine with swimming were less in trained rats than in controls. We conclude that exercise conditioning ameliorates hypertension in rats. The mechanism may involve an effect on cation transmembrane transport, as well as decreased, adrenosympathetic tone. Moreover, these effects may be related.

Vascular conversion of angiotensin I in stroke-prone spontaneously hypertensive and Wistar-Kyoto rats.

OBJECTIVE: Linkage studies have shown that the gene locus for angiotensin converting enzyme (ACE) is associated with the expression of hypertension in stroke-prone spontaneously hypertensive rats (SHRSP). We tested the hypothesis that the conversion of angiotensin I (Ang I) to angiotensin II (Ang II) in blood vessels is elevated in SHRSP. DESIGN: We measured the conversion rate of Ang I to Ang II during one pass through an isolated resistance vessel bed. We used the same substrains of SHRSP and Wistar-Kyoto control rats (WKY) that had been employed in the earlier linkage studies. METHODS: Isolated hindquarters from young and adult (10- to 12- and 36- to 38-week-old) rats were perfused with an artificial medium and then infused with Ang I at 0.5 and 2 pmol/ml. Ang I and II were measured with high-performance liquid chromatography and radioimmunoassay in hindquarter effluent and in blank control channels. Conversion and extraction rates were calculated from angiotensin levels in hindquarter and blank perfusion channels, respectively. RESULTS: The conversion rates of Ang I to Ang II did not differ between SHRSP and WKY in young or in adult rats. Captopril completely abolished the formation of Ang II in all groups of rats. During infusion at the higher dose of Ang I, the extraction of Ang I was significantly decreased in SHRSP compared with WKY. CONCLUSIONS: Our results are consistent with the notion that the metabolism of angiotensin is decreased in spontaneously hypertensive rats. However, we found no support for the hypothesis that vascular ACE is responsible for high blood pressure in SHRSP. These findings suggest that other genes close to the ACE locus or the hyperexpression of the enzyme in other areas may contribute to hypertension in these rats.

Increased potency of neuropeptide Y to antagonize alpha2-adrenoceptor function in the nucleus tractus solitarii of the spontaneously hypertensive rat.

The regulation by neuropeptide Y of alpha2-adrenoceptors in the nucleus tractus solitarii was evaluated in the adult normotensive Wistar Kyoto rat and the adult spontaneously hypertensive rat. The microinjection of a submaximal dose of l-noradrenaline (800 pmol in 50 nl) alone into the nucleus tractus solitarii produced a significant reduction in the mean arterial blood pressure in either strain. The threshold dose (1 pmol in 50 nl) of neuropeptide Y(1-36) for the vasodepressor response in the Wistar Kyoto rat was five times higher than that (0.2 pmol in 50 nl) in the spontaneously hypertensive rat. Furthermore, neuropeptide Y(1-36) at 0.2 pmol in 50 nl could significantly counteract the vasodepressor response to l-noradrenaline (800 pmol in 50 nl) in the spontaneously hypertensive rat, but not in the Wistar Kyoto rat, in which 1 pmol in 50 nl of neuropeptide Y(1-36) must be employed to counteract the vasodepressor response to l-noradrenaline (800 pmol in 50 nl), although the vasodepressor responses are of a similar magnitude. The in situ hybridization and quantitative receptor autoradiographical experiments showed that the alpha2A-adrenoceptor messenger RNA levels and the B(max) value of the alpha2-adrenoceptor agonist [3H]p-aminoclonidine binding sites measured in the nucleus tractus solitarii of the spontaneously hypertensive rat were substantially lower than those in the Wistar Kyoto rat. The quantitative receptor autoradiographical results were consistent with the cardiovascular results and showed that in the spontaneously hypertensive rat, neuropeptide Y(1-36) at 1 nM led to a significant increase in the K(d) value of [3H]p-aminoclonidine binding sites. In the Wistar Kyoto rat, neuropeptide Y(1-36) produced this effect only at 10 nM. The present study provides evidence for an increase of the potency of neuropeptide Y(1-36) to antagonistically modulate alpha2-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat. This enhanced antagonistic action may partly be related to a reduction in the number of alpha2A-adrenoceptors in the nucleus tractus solitarii of the spontaneously hypertensive rat, since a decrease has been observed in the alpha2A-adrenoceptor messenger RNA levels and the alpha2-adrenoceptor binding sites in the spontaneously hypertensive rat. This increased potency of neuropeptide Y(1-36) to antagonize alpha2-adrenoceptor function in the nucleus tractus solitarii of the spontaneously hypertensive rat may contribute to the development of high blood pressure in this hypertensive strain.

Antibiotics attenuate experimental hypertension in rats.

Hypertension was produced in Sprague-Dawley rats by intramuscular injections of either corticosterone or ACTH. Lower increases in blood pressure to these challenges were observed in Sprague-Dawley rats pretreated with neomycin or vancomycin which alone had no effect on blood pressure or growth. The development of high blood pressure in spontaneously hypertensive rats of a stroke-prone substrain was also attenuated by oral administration of neomycin. These results suggest that experimental hypertension can be modulated by the administration of antibiotics.

Effects of nifedipine and moxonidine on cardiac structure in spontaneously hypertensive rats. Stereological studies on myocytes, capillaries, arteries, and cardiac interstitium.

Light and electron microscopic stereological studies were performed on the myocardium of spontaneously hypertensive rats (SHR-SP) before and after treatment with nifedipine (27 mg/kg body weight/day) and the antisympathotonic agent moxonidine (8 mg/kg body weight/day). The treated groups were compared with nontreated SHR-SP and normotensive WKY (n = 10 in each group). At the beginning of therapy (when the male SHR-SP were 6 months old), blood pressure was increased and left ventricular hypertrophy had developed whereas pathologic changes of myocardial structure were not observed. After 3 months, the nontreated hypertensive rats showed cardiac fibrosis, activation and proliferation of interstitial cells, wall thickening of intramyocardial arteries, reduced capillarization as well as focal degeneration of myocytes at the ultrastructural level. Both treatments showed similar effects on blood pressure, degree of hypertrophy, and cardiac structure. Blood pressure as well as the degree of hypertrophy were significantly reduced. As far as myocardial fibrosis, capillarization, and regressive changes of myocytes are concerned a complete normalization was observed. Furthermore, nifedipine enhanced capillary supply beyond the normal level by induction of capillary neoformation. Microarteriopathy and activation of nonvascular interstitial cells (first step in development of interstitial myocardial fibrosis) were significantly suppressed by therapy, but the level of the normotensive control could not be maintained. Additional experiments with a low dose combination therapy of nifedipine and moxonidine that did not reduce blood pressure provided evidence that hypertension is an important determinant of the alterations of intramyocardial arteries, but not of cardiac interstitial fibrosis.

Sexual dimorphism of blood pressure: possible role of the renin-angiotensin system.

The prevalence of hypertension in men is higher than in women and the onset of this disease is earlier in male than in female subjects. In spontaneously hypertensive rats, males also have higher blood pressures than females. Evidence from epidemiological, physiological, molecular biological and morphological studies concerning this sexual dimorphism is reviewed. We demonstrate that the gonadal steroids testosterone and estrogen have important effects on the gene regulation of the renin-angiotensin system. This may in part contribute to the sexual dimorphism in blood pressure control. The direct effect of steroid hormones on genes related to hypertension provides a suitable paradigm to improve our understanding of molecular and cellular mechanisms of cardiovascular control.

Evidence for a differential modulation of the alpha-2 adrenoceptors by angiotensin II in the nucleus tractus solitarii of the spontaneously hypertensive and the Wistar-Kyoto normotensive rats.

An interaction between angiotensin II (Ang II) receptors and alpha 2-adrenoceptors was evaluated in the nucleus tractus solitarii (NTS) of the normotensive Wistar-Kyoto rat (WKY) and of the spontaneously hypertensive rat (SHR) using quantitative receptor autoradiography and cardiovascular analysis. In the WKY rat, Ang II promoted a dose-dependent increase in the IC50 value of l-noradrenaline when competing for ([3H]p-aminoclonidine ([3H]PAC) binding sites, which reached a maximum of 400% with 10 nM of Ang II and was associated with a small decrease in the B0 value (20%). In the SHR Ang II (0.1 nM) had an opposite effect leading to a decrease in the IC50 value of about 57%, and no change was observed in the B0 value. Saturation analysis also showed that Ang II (0.1 nM) increased the KD value of [3H]PAC in the WKY strain but in contrast decreased the KD value of [3H]PAC in the SHR. The Bmax value was not significantly changed neither in the WKY rat nor in the SHR. The cardiovascular analysis showed that a threshold dose of Ang II (0.05 pmol) counteracted the vasodepressor effect produced by l-noradrenaline coinjected in the NTS of the WKY rat. No effect was observed in heart rate. In the SHR no counteraction of the l-noradrenaline-induced vasodepressor effect was found, and in contrast a slight increase of the vasodepressor effect associated with a significant increase in the bradycardiac response was observed. The results give evidence for an antagonistic Ang II/alpha 2 receptor interaction in the cardiovascular part of the NTS of the WKY rat as previously observed in the Sprague-Dawley rat. However, this interaction is altered in the SHR, so that in this strain the Ang II/alpha 2 receptor interaction enhances alpha 2 affinity and possibly alpha 2 receptor function. This opposite effect observed in the SHR may represent one compensatory mechanism to counteract the development of high blood pressure in the SHR.

Regulation of calcitonin gene-related peptide mRNA expression in the hearts of spontaneously hypertensive rats by testosterone.

Previous studies have demonstrated the existence of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibres and nerve cell bodies in the rat heart. Using polymerase chain reaction we have investigated whether CGRP messenger RNA (mRNA) could be detected in heart tissue of spontaneously hypertensive rats, and whether CGRP-mRNA levels are affected by gonadectomy and testosterone substitution. Two weeks after castration CGRP-mRNA levels decreased to 65.2 +/- 6.4% of control values, whereas daily dihydrotestosterone substitution reversed this effect (88.0 +/- 1.2% of control). Our results indicate that steroid hormones control the expression of intracardiac CGRP on a pretranslational level.