Biomedical subjects

C D Sigmund

Publications and source records attributed to C D Sigmund.

At least 37 records · Page 2Linked to original sources

PubMed2000

Identification of cis elements in the cardiac troponin T gene conferring specific expression in cardiac muscle of transgenic mice.

To investigate the underlying mechanism regulating cardiac gene expression, transgenic mice carrying the rat cardiac troponin T proximal promoter (-497 bp from the transcriptional start site) fused to a LacZ or chloramphenicol acetyltransferase (CAT) reporter gene were analyzed. The LacZ expression pattern throughout development was very similar to that of the endogenous cardiac troponin T gene. Within this promoter, a high degree of sequence homology was found at 2 sites, modules D (-335 to -289 bp) and F (-249 to -209 bp). Both regions contain at least a TCTG(G/C) direct repeat and an A/T-rich site, whereas only the F module has a muscle enhancer factor 2 (MEF2)-like motif. No significant decrease in CAT transgene expression was observed when only the MEF2 core sequence was mutated. However, when the MEF2 core sequence and its flanking TCTGG site were mutated (Mut5), CAT transgene expression was significantly decreased in the heart, and ectopic expression of the transgene was also observed. When mutations were introduced into this promoter to destroy all upstream TCTG(G/C) direct repeats in the D module (MutD), CAT expression remained cardiac specific, but the expression level was dramatically decreased. Relaxation of cardiac-specific transgene expression became even more severe in transgenic mice carrying double mutations (Mut[D+5]). In addition, CAT activity in the heart was nearly abolished. These results suggest that D and F modules have an additive function in determining the level of expression in the heart and only the F module confers cardiac-specific expression.

Animals↗

PubMed2000

Transgenic models as tools for studying the regulation of human renin expression.

Transgenic mice and rats have become popular tools to study the regulation of gene expression and the consequences of protein over-production. Over the past decade, numerous transgenic models have been developed to study the mechanisms of human renin gene expression and the participation of the renin-angiotensin system in the development of hypertension. Herein we will provide an overview of what has been learned from the use of transgenic models for studying the human renin gene.

Animals↗

PubMed2000

Appropriate tissue- and cell-specific expression of a single copy human angiotensinogen transgene specifically targeted upstream of the HPRT locus by homologous recombination.

Development of experimental models by genetic manipulation in mice has proven to be very useful in determining the significance of particular genes in the development of or susceptibility to hypertension. Advances in molecular genetics, transgenic mouse technology, and physiological measurements in mice provided an opportunity to go a step further and develop models to analyze the physiological significance of specific gene variants potentially causing hypertension. In this report, we describe the development of a human angiotensinogen transgenic mouse model generated by targeting the human angiotensinogen gene upstream of the mouse HPRT locus by homologous recombination. The main benefit of this transgenic mouse model is that the human angiotensinogen gene is inserted into the mouse genome as a single copy at a predefined locus and in a specific orientation-a process that can be repeated utilizing other variants of this gene. We establish the validity of this approach by showing that the hAGT(hprt) mice have normal tissue- and cell-specific expression of the human angiotensinogen gene and normally produce and process the hAGT protein at physiological levels.

Angiotensinogen↗

PubMed2000

Transgenic and knockout mice to study the renin-angiotensin system and other interacting vasoactive pathways.

Essential hypertension is an insidious disease in which the afflicted person risks disability and death from myocardial infarction and stroke. Many factors contribute to the development of essential hypertension, including environment, diet, daily stress, and genetics. Although several single gene disorders causing high blood pressure have been identified, the genetics of essential hypertension are much more complicated. The current hypothesis is that a combination of genetic variations in multiple genes may predispose a person to hypertension. Both overexpression and gene inactivation ("knockout") have proven useful tools to evaluate the genetics of essential hypertension and to identify pathways regulating blood pressure. Molecular and physiologic evaluations of transgenic and knockout mice carried out over the past 5 years have provided a plethora of information about the mechanisms of blood pressure regulation and the development and maintenance of hypertension. This review focuses on the newer mouse models that have been developed to investigate hypertension with an emphasis on vascular and renal mechanisms, contributed by the renin-angiotensin system, and other pathways intersecting with the renin-angiotensin system.

Angiotensin II↗

PubMed2000

Understanding hypertension through genetic manipulation in mice.

With the advances in mouse molecular genetics and physiology during the last decade, the mouse has become the animal model of choice for studying the genetic basis of many diseases. Terms such as "transgenic" and "knockout" have become part of a colloquial language used in most research laboratories that are investigating human diseases. These terms refer to the two most commonly used methods for analyzing the function of a gene in vivo: overexpression (transgenic mouse) and deletion (knockout mouse). Both methods have proved to be extremely useful in establishing the importance of specific genes in genetic disorders, such as hypertension. The choice of genes being investigated in relationship to hypertension was governed by the knowledge of systems regulating vascular and renal physiology. Thus, it is not surprising that most of the focus was given to the renin-angiotensin system (RAS). Apart from the RAS, other systems known to regulate vascular tone and/or electrolyte and fluid homeostasis have also been analyzed using transgenic and knockout approaches. This review briefly summarizes some of the mouse models relevant to renal mechanisms of hypertension and then discusses the future of genetic manipulation in mice for studying the genetics of hypertension.

Animals↗

PubMed2000

Viewpoint: are studies in genetically altered mice out of control?

Because the use of transgenic and gene-targeted models has increased in popularity, the number of reports describing unpredictable phenotypic effects caused by variation in the genetic background used to generate or propagate these models has steadily increased. There are now many examples in which animals containing the same exact genetic manipulation exhibit profoundly different phenotypes when present on diverse genetic backgrounds, demonstrating that genes unrelated, per se, to the ones being targeted can play a significant role in the observed phenotype. Herein, I will discuss (1) the source of genetic variability in mutant mouse models, (2) the appropriateness of using inbred mice as controls, and (3) strategies to help minimize genetic variation between experimental and control mice.

Animals↗

PubMed2000

Impaired endothelial function in transgenic mice expressing both human renin and human angiotensinogen.

BACKGROUND AND PURPOSE: Chronic hypertension is a risk factor for carotid vascular disease and stroke. Mechanisms that account for alterations in carotid and cerebral vascular function during hypertension are poorly defined and based almost exclusively on studies in the spontaneously hypertensive rat, a model in which hypertension has an unknown etiology and in which the genetic background is dissimilar to the most commonly used normotensive control, the Wistar-Kyoto rat. METHODS: In this study we examined vascular function in a defined model of hypertension, double transgenic mice that overexpress both human renin (R+) and human angiotensinogen (A+). We studied vessels in vitro from R+/A+ mice as well as nontransgenic (R-/A-) and single transgenic (R-/A+ or R+/A-) littermate controls. RESULTS: After submaximal precontraction with U46619 or prostaglandin F(2alpha), acetylcholine, which produces relaxation mediated by endothelial nitric oxide synthase, produced marked relaxation of carotid arteries in control mice but was impaired in R+/A+ mice. For example, 1 micromol/L acetylcholine relaxed the carotid artery by 79+/-4% versus 44+/-7% (P<0.01) in control and R+/A+ mice, respectively. Impaired responses to acetylcholine in R+/A+ mice could be restored toward normal with indomethacin (10 micromol/L). In contrast, relaxation of the carotid artery in response to nitroprusside and papaverine was similar in R+/A+ mice and control mice. CONCLUSIONS: These findings indicate that acetylcholine-induced relaxation of carotid artery is impaired selectively in mice made hypertensive by expression of human renin and human angiotensinogen. The mechanism of this impairment may involve production of a cyclooxygenase-derived contracting factor.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗

PubMed2000

Divergent functions of angiotensin II receptor isoforms in the brain.

The renin-angiotensin system (RAS) plays a critical role in cardiovascular and fluid homeostasis. The major biologically active peptide of the RAS is angiotensin II, which acts through G protein-coupled receptors of two pharmacological classes, AT(1) and AT(2). AT(1) receptors, expressed in brain and peripheral tissues, mediate most classically recognized actions of the RAS, including blood pressure homeostasis and regulation of drinking and water balance. In rodents, two highly homologous AT(1) receptor isoforms, termed AT(1A) and AT(1B) receptors, are expressed at different levels in major forebrain cardiovascular and fluid regulatory centers, with AT(1A) expression generally exceeding AT(1B) expression, but the relative contributions of these receptor subtypes to central angiotensin II responses are not known. We used gene targeting in combination with a unique system for maintaining catheters in the cerebral ventricles of conscious mice to test whether there are differential roles for AT(1A) and AT(1B) receptors in responses elicited by angiotensin II in the brain. Here we show that the blood pressure increase elicited by centrally administered angiotensin II can be selectively ascribed to the AT(1A) receptor. However, the drinking response requires the presence of AT(1B) receptors. To our knowledge, this is the first demonstration of a primary and nonredundant physiological function for AT(1B) receptors.

Angiotensin II↗

PubMed1999

Highly regulated cell type-restricted expression of human renin in mice containing 140- or 160-kilobase pair P1 phage artificial chromosome transgenes.

We generated transgenic mice with two P1 artificial chromosomes, each containing the human renin (HREN) gene and extending to -35 and -75 kilobase pairs, respectively. HREN protein production was restricted to juxtaglomerular cells of the kidney, and its expression was tightly regulated by angiotensin II and sodium. The magnitude of the up- and down-regulation in HREN mRNA caused by the stimuli tested was identical to the endogenous renin gene, suggesting tight physiological regulation. P1 artificial chromosome mice were mated with transgenic mice overexpressing human angiotensinogen to determine if there was a chronic compensatory down-regulation of the transgene. Despite a 3-fold down-regulation of HREN mRNA, plasma angiotensin II and blood pressure was modestly elevated in the double transgenic mice. Nevertheless, this elevation was significantly less than a different double transgenic model containing a poorly regulated HREN transgene. The increase in blood pressure, despite the decrease in HREN mRNA, suggests that the HREN gene can partially, but not completely, compensate for excess circulating angiotensinogen. These data suggest the possibility that increases in circulating or tissue angiotensinogen may cause an increase in blood pressure in humans, even in the presence of a functionally active servo-mechanism to down-regulate HREN expression.

Angiotensin II↗

PubMed1999

Species-specific differences in positive and negative regulatory elements in the renin gene enhancer.

A distal transcriptional enhancer has been previously reported upstream of the mouse renin gene. A homologous sequence is also present upstream of the human renin gene, but the mouse and human renin enhancers differ markedly in their ability to activate transcription of a renin promoter. Although the 2 enhancers share high homology in their 202-bp promoter distal portions, their 40-bp proximal portions are heterogeneous. Chimeric enhancers were used to test the role of the 40-bp segment (m40) of the enhancer by using transient transfection analysis in mouse kidney renin-expressing As4. 1 cells. Deletion of m40 from the mouse renin enhancer or its addition to the human renin enhancer did not significantly change transcriptional activity when placed close to a mouse or human renin promoter. However, when placed further upstream of a renin promoter, the same deletion and substitution markedly altered enhancer activity. Electrophoretic gel mobility shift analysis identified 2 elements, a and b, in m40 that specifically bound nuclear proteins from As4.1 cells. Mutagenesis and transient transfection analysis revealed that element b accounts for the function of m40 and that element a antagonizes the positive influence of element b. Gel competition and supershift analysis revealed that nuclear factor-Y, a ubiquitous CAAT-box binding protein, binds to element a. Sequence analysis revealed that the human renin enhancer has a natural loss-of-function mutation in element b that affects its ability to transactivate when placed far upstream of a promoter. Reversion of the human renin element b to match the mouse sequence restored transactivation of the enhancer in mouse As4.1 cells. These data suggest that element b cooperates with the rest of the enhancer to maintain full enhancer activity, whereas element a may regulate enhancer activity. Sequence differences in these elements may explain the functional differences in the mouse and human renin enhancer sequences.

Animals↗

PubMed1999

Efficient liver-specific deletion of a floxed human angiotensinogen transgene by adenoviral delivery of Cre recombinase in vivo.

Tissue-specific ablation of gene function is possible in vivo by the Cre-loxP recombinase system. We generated transgenic mice containing a human angiotensinogen gene flanked by loxP sites (hAGT(flox)). To examine the physiologic consequences of tissue-specific loss of angiotensinogen gene function in vivo, we constructed an adenovirus expressing Cre recombinase. Studies were performed in several independent lines of hAGT(flox) mice before and after intravenous administration of either Adcre or AdbetaGal as a control. Systemic administration of Adcre caused a significant decrease in circulating human angiotensinogen and markedly blunted the pressor response to administration of purified recombinant human renin. Southern blot analysis of genomic DNA from various organs revealed that the Cre-mediated deletion was liver-specific. Further analysis revealed the absence of full-length human angiotensinogen mRNA and protein in the liver but not the kidney of Adcre mice, consistent with the liver being the target for adenoviruses administered intravenously. These studies demonstrate that extra-hepatic sources of angiotensinogen do not contribute significantly to the circulating pool of angiotensinogen and provide proof-of-principle that the Cre-loxP system can be used effectively to examine the contribution of the systemic and tissue renin-angiotensin system to normal and pathological regulation of blood pressure.

Adenoviridae↗

PubMed1999

Novel mechanism of hypertension revealed by cell-specific targeting of human angiotensinogen in transgenic mice.

We tested the hypothesis that the tissue-specific intrarenal renin-angiotensin system (RAS) can participate in the regulation of blood pressure independently of its endocrine counterpart, by generating two transgenic models that differ in their tissue-specific expression of human angiotensinogen (AGT). Human AGT expression was driven by its endogenous promoter in the systemic model and by the kidney androgen-regulated protein promoter in the kidney-specific model. Using molecular, biochemical, and physiological measurements, we demonstrate that human AGT mRNA and protein are restricted to the kidney in the kidney-specific model. Plasma ANG II was elevated in the systemic model but not in the kidney-specific model. Nevertheless, blood pressure was markedly elevated in both the systemic and kidney-specific transgenic mice. Acute administration of the selective ANG II AT-1 receptor antagonist losartan lowered blood pressure in the systemic model but not in the kidney-specific model. These results provide evidence for the potential importance of the intrarenal RAS in blood pressure regulation by showing that expression of AGT specifically in the kidney leads to chronic hypertension independently of the endocrine RAS.

Angiotensin Receptor Antagonists↗

PubMed1999

The angiotensinogen gene is expressed in both astrocytes and neurons in murine central nervous system.

Two transgenic mouse models were used to examine the cellular localization of angiotensinogen (AGT) in the brain. The first model was previously described in detail and consists of a human AGT genomic transgene containing all exons and introns of the gene and 1. 2 kb of the 5' flanking DNA. The second model contains a fusion between 1.2 kb of HAGT 5' flanking DNA and the beta-gal reporter gene which exhibits a similar pattern of tissue-specific expression to the HAGT transgene. Expression of both transgenes qualitatively mirrors the expression of endogenous AGT. Double staining of transgenic mouse brain sections with X-gal and GFAP revealed that a majority of beta-gal activity was localized to astrocytes in almost all brain areas. However, both beta-gal activity as identified by X-gal, and HAGT mRNA as detected by in situ hybridization, were also found in neurons in restricted areas of the brain, including the mesencephalic trigeminal nucleus (meV), subfornical organ (SFO) and the external lateral parabrachial nucleus (elPB). The expression of these transgenes provides the first convincing evidence for AGT gene expression in neurons in the brain. We further report by angiotensin II (Ang-II) immunostaining in rat brains after selective lesioning, that Ang-II is likely involved in a neuronal pathway from the PB to the amygdala (Ce). Finally, we performed double-labeling, first by retrograde labeling of HRP injected into the Ce, and then by X-gal on PB neurons in beta-gal transgenic mice, and identified doubly labeled neurons. Based on these results, we propose that AGT is generated in neurons in the elPB, transported to the Ce and converted into Ang-II locally to exert is biological functions.

Angiotensinogen↗

PubMed1999

Contrasting blood pressure effects of obesity in leptin-deficient ob/ob mice and agouti yellow obese mice.

OBJECTIVE: Recent advances in understanding the neuroendocrine pathways regulating appetite, metabolism and body weight afford an opportunity to explore further the mechanisms by which obesity influences arterial pressure. ob/ob(Lep(ob)/Lep(ob)) mice have a mutation in the ob gene and are leptin-deficient. Leptin possesses pressor actions and has been shown to increase arterial pressure when infused chronically or over-expressed transgenically. In contrast, agouti yellow obese(Ay) mice have overexpression of an agouti peptide that blocks melanocortin receptors. Stimulation of melanocortin receptors by alpha-melanocyte-stimulating hormone decreases arterial pressure. DESIGN AND METHODS: This study measured arterial pressure in leptin-deficient ob/ob mice, agouti yellow obese mice and their lean controls to test the hypothesis that the effects of obesity on arterial pressure are importantly influenced by the genetic and neuroendocrine mechanisms causing the obesity. We measured arterial pressure directly in conscious ob/ob mice (n = 14), agouti yellow obese mice (n = 6) and the same number of lean littermates. RESULTS: Body weight was nearly twice as high in ob/ob mice as in their lean controls, but mean arterial pressure was significantly lower in ob/ob mice (92+/-3 mmHg) compared with their lean controls (106+/-2 mmHg; P = 0.00017). In contrast, mean arterial pressure was significantly higher in agouti yellow obese mice (124+/-3 mmHg) than in their lean controls (99+/-1 mmHg; P = 0.000002) despite the fact that the agouti mice had milder obesity. CONCLUSIONS: This study prompts three conclusions: (1) leptin-deficient ob/ob mice and agouti yellow obese mice have contrasting blood pressure responses to obesity, (2) obesity does not invariably increase arterial pressure in mice, and (3) the arterial pressure response to obesity may depend critically on the underlying genetic and neuroendocrine mechanisms.

Animals↗

PubMed1999

Gene transfer of endothelial nitric oxide synthase (eNOS) in eNOS-deficient mice.

Relaxation to acetylcholine (ACh) and calcium ionophore (A-23187) is absent in aortas from endothelial nitric oxide synthase (eNOS)-deficient (eNOS -/-) mice. We hypothesized that gene transfer of eNOS would restore relaxation to ACh and A-23187 in eNOS -/- mice. Aortic rings from eNOS -/- and eNOS +/+ mice were exposed in vitro to vehicle or adenoviral vectors encoding beta-galactosidase (lacZ) or eNOS. Histochemical staining for beta-galactosidase and eNOS demonstrated transduction of endothelial cells and adventitia. Vehicle-treated vessels from eNOS -/- mice did not relax to ACh or A-23187 compared with eNOS +/+ mice. In contrast, relaxation to nitroprusside (NP) was significantly greater in eNOS -/- mice than in eNOS +/+ mice. Gene transfer of eNOS, but not lacZ, to vascular rings of eNOS -/- mice restored relaxation to ACh and A-23187. In vessels from eNOS -/- mice that were transduced with eNOS, N(omega)-nitro-L-arginine (10(-4) M) inhibited relaxation to ACh and A-23187 but not NP. Thus vascular function can be significantly improved by gene transfer in vessels where a major relaxation mechanism is genetically absent.

Adenoviridae↗

PubMed1999

JG cell expression and partial regulation of a human renin genomic transgene driven by a minimal renin promoter.

In the kidney, renin gene expression is exquisitely localized to the juxtaglomerular (JG) cells lining the afferent arteriole, having the capacity to regulate renin synthesis in response to a variety of physiological cues. We investigated human renin gene expression in transgenic mice containing a genomic construct driven by 149 bp of its proximal promoter to elucidate whether this was sufficient to confer JG-specific expression. Whereas human renin mRNA was permissively expressed in most tissues, the transgene was expressed mainly in JG cells in the kidney. Active human renin and human prorenin were found in the systemic circulation at levels consistent with previous transgenic models. Remarkably, two lines displayed an appropriate upregulation of transgene mRNA in response to angiotensin-converting enzyme inhibition, and two lines exhibited a downregulation of transgene mRNA in response to subpressor and pressor doses of ANG II. Our results suggest that 149 bp of the human renin proximal promoter, in a context of a genomic construct, are sufficient to confer human renin expression in renal JG cells and at least some aspects of appropriate regulation.

Angiotensin II↗

PubMed1999

Human renin mRNA stability is increased in response to cAMP in Calu-6 cells.

The human carcinoma-derived cell line Calu-6 has previously been demonstrated to endogenously express human renin (hREN) mRNA and to markedly increase steady-state hREN mRNA levels (100-fold after 24 hours) in response to analogues of cAMP and postreceptor activators of adenylyl cyclase such as forskolin. However, both transfection analysis using hREN promoter-reporter constructs and nuclear run-on experiments suggest that transcriptional activity alone cannot account for this level of induction. We performed primer extension, reverse transcription-polymerase chain reaction, and 3' rapid amplification of cDNA ends to compare hREN mRNA between unstimulated and forskolin-stimulated cells. We demonstrate that hREN mRNA is identical under both conditions with respect to (1) utilization of the appropriate transcription start site, (2) processing of renin mRNA, and (3) utilization of the proper polyadenylation site and length of the poly-A tail. To address the mechanism of induction caused by cAMP, we used transcriptional inhibition and measured decay of hREN mRNA before and after forskolin or phorbol ester treatment. Experiments with both actinomycin D and 5, 6-dichlororibofuranosylbenzimidazole (DRB) showed that forskolin treatment markedly stabilized hREN mRNA in Calu-6 cells. A 2.3-fold increase in hREN mRNA half-life was also observed after treatment of Calu-6 cells with phorbol ester. Experiments with DRB demonstrated a similar robust stabilization of hREN mRNA after forskolin and phorbol ester treatment. These data demonstrate that the induction in hREN mRNA in response to both cAMP and phorbol ester occurs by a mechanism involving a posttranscriptional component.

Colforsin↗

PubMed1998

The brain renin-angiotensin system contributes to the hypertension in mice containing both the human renin and human angiotensinogen transgenes.

We have previously shown that mice transgenic for both the human renin and human angiotensinogen genes (RA+) exhibit appropriate tissue- and cell-specific expression of both transgenes, have 4-fold higher plasma angiotensin II (AII) levels, and are chronically hypertensive. However, the relative contribution of circulating and tissue-derived AII in causing hypertension in these animals is not known. We hypothesized that the brain renin-angiotensin system contributes to the elevated blood pressure in this model. To address this hypothesis, mean arterial pressure (MAP) and heart rate were measured in conscious, unrestrained mice after they were instrumented with intracerebroventricular cannulae and carotid arterial and jugular vein catheters. Intracerebroventricular administration of the selective AII type 1 (AT-1) receptor antagonist losartan (10 microgram, 1 microL) caused a significantly greater peak fall in MAP in RA+ mice than in nontransgenic RA- controls (-29+/-4 versus -4+/-2 mm Hg, P<0.01). To explore the mechanism of a central renin-angiotensin system-dependent hypertension in RA+ mice, we determined the relative depressor responses to intravenous administration of the ganglionic blocking agent hexamethonium (5 mg/kg) or an arginine vasopressin (AVP) V1 receptor antagonist (AVPX, 10 microgram/kg). Hexamethonium caused equal lowering of MAP in RA+ mice and controls (-46+/-3 versus -52+/-3, P>0.05), whereas AVPX caused a significantly greater fall in MAP in RA+ compared with RA- mice (-24+/-2 versus -6+/-1, P<0.01). Consistent with this was the observation that circulating AVP was 3-fold higher in RA+ mice than in control mice. These results suggest that increased activation of central AT-1 receptors, perhaps those located at sites involved in AVP release from the posterior pituitary gland, plays a role in the hypertension in RA+ mice. Furthermore, our finding that both human transgenes are expressed in brain regions of RA+ mice known to be involved in cardiovascular regulation raises the possibility that augmented local production of AII and increased activation of AT-1 receptors at these sites is involved.

Angiotensins↗