Gillian Brooker

Targeted Inactivation of the Ren-2 Gene in Mice

Several recent studies have demonstrated that ablation of genes of the renin-angiotensin system can have wide-ranging and sometimes unexpected effects. Renin is directly involved in blood pressure regulation and is encoded by a single gene in most mammals. Wild mouse strains and some inbred laboratory strains have a duplicated renin gene (Ren-2), the physiological significance of which is unclear. Significant differences exist in the structure and expression of these renin genes, but as yet, no distinct biological function that distinguishes these genes has been defined. We have used gene targeting to discover the effects of inactivating the duplicated (Ren-2) gene in strain 129 mice, and we show that mice lacking the Ren-2 gene are viable and healthy. There appear to be no histopathological differences in renin-expressing tissues between Ren-2-null mice and their controls. Studies of our Ren-2-null mice allow, for the first time, a direct evaluation of the ability of the Ren-1d gene to regulate blood pressure in the absence of expression of the Ren-2 enzyme. We observed no alteration to blood pressure in adult mice homozygous for the mutated Ren-2 gene, even though the concentration of active renin is increased and of prorenin is decreased in plasma of these mice. Ren-1d is therefore capable of regulating normal blood pressure and despite a different tissue expression profile, is functionally equivalent to Ren-1c.

Circadian control of mouse heart rate and blood pressure by the suprachiasmatic nuclei: behavioral effects are more significant than direct outputs

Background Diurnal variations in the incidence of events such as heart attack and stroke suggest a role for circadian rhythms in the etiology of cardiovascular disease. The aim of this study was to assess the influence of the suprachiasmatic nucleus (SCN) circadian clock on cardiovascular function. Methodology/Principal Findings Heart rate (HR), blood pressure (BP) and locomotor activity (LA) were measured in circadian mutant (Vipr2 −/−) mice and wild type littermates, using implanted radio-telemetry devices. Sleep and wakefulness were studied in similar mice implanted with electroencephalograph (EEG) electrodes. There was less diurnal variation in the frequency and duration of bouts of rest/activity and sleep/wake in Vipr2 −/− mice than in wild type (WT) and short “ultradian” episodes of arousal were more prominent, especially in constant conditions (DD). Activity was an important determinant of circadian variation in BP and HR in animals of both genotypes; altered timing of episodes of activity and rest (as well as sleep and wakefulness) across the day accounted for most of the difference between Vipr2 −/− mice and WT. However, there was also a modest circadian rhythm of resting HR and BP that was independent of LA. Conclusions/Significance If appropriate methods of analysis are used that take into account sleep and locomotor activity level, mice are a good model for understanding the contribution of circadian timing to cardiovascular function. Future studies of the influence of sleep and wakefulness on cardiovascular physiology may help to explain accumulating evidence linking disrupted sleep with cardiovascular disease in man.

αV Integrins Are Necessary for Eutrophic Inward Remodeling of Small Arteries in Hypertension

Human essential hypertension is characterized by eutrophic remodeling of small arteries, with little evidence of hypertrophy. Likewise, vessels of young hypertensive TGR(mRen2)27 animals have undergone similar structural alterations. The role of integrins in resistance arteries of TGR(mRen2)27 during the eutrophic-remodeling process was examined as blood pressure rose. Initially, 8 &agr; and 3 &bgr; integrins were identified and levels of expression investigated using RT-PCR. As pressure increased and remodeling advanced, integrin expression profiles revealed that only &agr;V was significantly raised. In conjunction, we confirmed elevated integrin &agr;V protein levels in TGR(mRen2)27 rat arteries and localization to the media using immunofluorescence. &bgr;1 and &bgr;3, but not &bgr;5 integrin subunits were coprecipitated with integrin &agr;V and are implicated in the eutrophic remodeling process. Administration of a peptide antagonist of &agr;V&bgr;3 abolished remodeling but enhanced growth, indicating that hypertrophy supervened as a response to hypertension-induced increases in wall stress. We have established that the only upregulated integrin, the &agr;V subunit of integrin &agr;V&bgr;3, has a crucial role in the hypertensive remodeling process of TGR(mRen2)27 rat resistance arteries. During hypertensive remodeling, functions of specific &agr;V&bgr;3-extracellular matrix interactions are likely to allow vascular smooth muscle cell–length autoregulation, which includes a migratory process, to maintain a narrowed lumen after a prolonged constricted state.

Real-Time Visualization of Human Prolactin Alternate Promoter Usage in Vivo Using a Double-Transgenic Rat Model

We have generated a humanized double-reporter transgenic rat for whole-body in vivo imaging of endocrine gene expression, using the human prolactin (PRL) gene locus as a physiologically important endocrine model system. The approach combines the advantages of bacterial artificial chromosome recombineering to report appropriate regulation of gene expression by distant elements, with double reporter activity for the study of highly dynamic promoter regulation in vivo and ex vivo. We show first that this rat transgenic model allows quantitative in vivo imaging of gene expression in the pituitary gland, allowing the study of pulsatile dynamic activity of the PRL promoter in normal endocrine cells in different physiological states. Using the dual reporters in combination, dramatic and unexpected changes in PRL expression were observed after inflammatory challenge. Expression of PRL was shown by RT-PCR to be driven by activation of the alternative upstream extrapituitary promoter and flow cytometry analysis pointed at diverse immune cells expressing the reporter gene. These studies demonstrate the effective use of this type of model for molecular physiology and illustrate the potential for providing novel insight into human gene expression using a heterologous system.

Angiotensin-converting Enzyme Is a Modifier of Hypertensive End Organ Damage

Severe forms of hypertension are characterized by high blood pressure combined with end organ damage. Through the development and refinement of a transgenic rat model of malignant hypertension incorporating the mouse renin gene, we previously identified a quantitative trait locus on chromosome 10, which affects malignant hypertension severity and morbidity. We next generated an inducible malignant hypertensive model where the timing, severity, and duration of hypertension was placed under the control of the researcher, allowing development of and recovery from end organ damage to be investigated. We have now generated novel consomic Lewis and Fischer rat strains with inducible hypertension and additional strains that are reciprocally congenic for the refined chromosome 10 quantitative trait locus. We have captured a modifier of end organ damage within the congenic region and, using a range of bioinformatic, biochemical and molecular biological techniques, have identified angiotensin-converting enzyme as the modifier of hypertension-induced tissue microvascular injury. Reciprocal differences between angiotensin-converting enzyme and the anti-inflammatory tetrapeptide, N-acetyl-Ser-Asp-Lys-Pro in the kidney, a tissue susceptible to end organ damage, suggest a mechanism for the amelioration of hypertension-dependent damage.