Chandi Griffin

Expression of AT2 receptors in the developing rat fetus.

Angiotensin II is known primarily for its effects on blood pressure and electrolyte homeostasis, but recent studies suggest that angiotensin II may play a role in the regulation of cellular growth. This study was undertaken to identify the angiotensin II receptor subtypes expressed during fetal and neonatal development and to characterize their cellular localization. Using an in situ receptor binding assay on sagittal frozen sections of fetal and neonatal rats, bound 125I-[Sar1,Ile8]-angiotensin II was visualized by film and emulsion autoradiography. Bound radioligand was detected by E11 (embryonic day 11) and maximal binding occurred by E19-21. Radioligand binding remained unaltered 30 min after birth, whereas a noticeable and stable decrease was observed 12 h postparturition. The highly abundant angiotensin II receptors were shown to be AT2 by the marked reduction in radioligand binding achieved with PD123177 (10(-7)M), a specific AT2 receptor antagonist, whereas DuP 753 (10(-5)M), an AT1 receptor antagonist, had little effect. Emulsion autoradiography showed radioligand binding in the undifferentiated mesenchyme of the submucosal layers of the intestine and stomach, connective tissue and choroid surrounding the retina, subdermal mesenchyme adjacent to developing cartilage, diaphragm, and tongue. Residual AT2 receptors were found on the dorsal subdermal region of the tongue 72 h after birth. AT1 receptors were detected in the placenta at E13 and in the aorta, kidney, lung, liver, and adrenal gland at E19-21, consistent with an adult distribution. The transient expression of AT2 receptors in the mesenchyme of the fetus suggests a role of angiotensin II in fetal development.

Integrated Array-Comparative Genomic Hybridization and Expression Array Profiles Identify Clinically Relevant Molecular Subtypes of Glioblastoma

Glioblastoma, the most aggressive primary brain tumor in humans, exhibits a large degree of molecular heterogeneity. Understanding the molecular pathology of a tumor and its linkage to behavior is an important foundation for developing and evaluating approaches to clinical management. Here we integrate array-comparative genomic hybridization and array-based gene expression profiles to identify relationships between DNA copy number aberrations, gene expression alterations, and survival in 34 patients with glioblastoma. Unsupervised clustering on either profile resulted in similar groups of patients, and groups defined by either method were associated with survival. The high concordance between these separate molecular classifications suggested a strong association between alterations on the DNA and RNA levels. We therefore investigated relationships between DNA copy number and gene expression changes. Loss of chromosome 10, a predominant genetic change, was associated not only with changes in the expression of genes located on chromosome 10 but also with genome-wide differences in gene expression. We found that CHI3L1/YKL-40 was significantly associated with both chromosome 10 copy number loss and poorer survival. Immortalized human astrocytes stably transfected with CHI3L1/YKL-40 exhibited changes in gene expression similar to patterns observed in human tumors and conferred radioresistance and increased invasion in vitro. Taken together, the results indicate that integrating DNA and mRNA-based tumor profiles offers the potential for a clinically relevant classification more robust than either method alone and provides a basis for identifying genes important in glioma pathogenesis.

The Cardiac Renin-Angiotensin System in STZ-Induced Diabetes

To determine the effect of diabetes on the cardiac renin-angiotensin system, we compared angiotensin II binding density and renin, angiotensinogen, and type 1 angiotensin II (AT1) receptor mRNA levels in hearts of Sprague-Dawley rats 14 days after the administration of streptozotocin (STZ), in vehicle-treated control rats, and in STZ-administered rats made euglycemic with insulin. Myocardial angiotensin II receptor density, determined using an in situ autoradiographic technique, was increased significantly in hyperglycemic diabetic rats in comparison with control rats and euglycemic diabetic rats (P < 0.01) as a result of an increase in both AT1 and AT2 (type 2 angiotensin II) subtypes. The myocardial AT1 receptor mRNA level, determined by slot blot hybridization, was also significantly greater in the hyperglycemic diabetic rats (P < 0.005). Neither plasma renin concentration nor cardiac renin or angiotensinogen mRNA levels differed among the three study groups. In an additional experiment, control and diabetic rats were infused with angiotensin II (200 ng · kg−1 · min−1 i.p. for 7 days) or vehicle. Plasma renin concentration decreased significantly, whereas no significant changes occurred in cardiac renin or angiotensinogen steady-state mRNA levels. As in the first experiment, levels of AT1 receptor mRNA were significantly greater in the diabetic rats. Thus, myocardial angiotensin II receptor density is increased in diabetic rats in association with an increase in steady-state AT1 receptor mRNA levels, an abnormality that appears to be independent of changes in the circulating renin-angiotensin system.

Developmental and hormonal regulation of glucocorticoid receptor messenger RNA in the rat.

The biological action of glucocorticoids is dependent upon tissue-specific levels of the glucocorticoid receptor (GR). During stress, the hypothalamic-pituitary-adrenal axis is stimulated, and high levels of glucocorticoids circulate. This axis is modulated by negative feedback by glucocorticoids, which inhibit hypothalamic and pituitary hormone secretion and downregulate GR gene expression. To study the developmental tissue-specific regulation of the GR, we measured the relative concentration of GR mRNA in fetal, neonatal, adult, and aged rats and examined the effects of dexamethasone on GR gene expression. Three different tissue-specific developmental patterns of GR mRNA accumulation were found. In addition, there was an age-dependent tissue-specific pattern in the feedback regulation of GR mRNA by glucocorticoids. In the fetus and neonate, GR mRNA abundance was not regulated by circulating glucocorticoids. The adult pattern of glucocorticoid feedback inhibition of GR mRNA expression appeared between 2 and 7 d of life in liver, and after 7 but before 14 d of age in brain. The GR was biologically active in the 2-d-old neonate, however, since dexamethasone enhanced gene expression of angiotensinogen, which is another glucocorticoid responsive gene. These data demonstrate that the GR gene is regulated by both developmental and tissue-specific factors, and provide another molecular basis for ontogenic variations in the hypothalamic-pituitary-adrenal response to stress.

Tissue-Specific Regulation of Type 1 Angiotensin II Receptor mRNA Levels in the Rat

Most of the biological effects of the renin-angiotensin system are mediated by the binding of angiotensin II (Ang II) to the type 1 Ang II (AT1) receptor, the predominant receptor subtype present after fetal life. To study tissue-specific regulation of the expression of the AT1 receptor in the rat, we altered activity of the renin-angiotensin system by feeding rats a low (0.07% NaCl), normal (0.3% NaCl), or high (7.5% NaCl) salt chow for 14 days; infusing Ang II (200 ng/kg per minute IP) or vehicle for 7 days; and administering an angiotensin-converting enzyme inhibitor (captopril, 100 mg/dL in the drinking water) or vehicle for 7 days. Renin, angiotensinogen, and total AT1 receptor mRNA levels were measured by slot-blot hybridization with cRNA probes, and AT1 receptor subtypes (A and B) were measured by reverse transcription-polymerase chain reaction in the presence of a cRNA internal standard. Plasma renin concentration and renal renin, renal and hepatic angiotensinogen, and hepatic AT1 receptor mRNA levels were all inversely related to salt intake; in contrast, renal AT1 receptor mRNA levels were significantly lower in rats fed low salt, a difference that was exclusively due to a decrease in the AT1A subtype. This difference did not appear to be mediated by a change in the circulating levels of Ang II, because Ang II infusion reduced plasma renin concentration and renal renin mRNA with no effect on either angiotensinogen or AT1 receptor mRNA levels in kidney or liver, renal Ang II receptor density (determined by in situ autoradiography) decreased, presumably via a posttranscriptional mechanism. Similarly, inhibition of Ang II generation with captopril increased plasma renin concentration and renal renin mRNA levels without altering renal or hepatic angiotensinogen mRNA or renal AT1 receptor mRNA levels. Thus, AT1 receptor gene expression is regulated in a tissue-specific manner that is distinct from other components of systemic and local renin-angiotensin systems and that appears to be mediated by a mechanism other than through changes in the circulating levels of Ang II.

Estrogen Receptor β Binds to and Regulates Three Distinct Classes of Target Genes

Estrogen receptor β (ERβ) has potent antiproliferative and anti-inflammatory properties, suggesting that ERβ-selective agonists might be a new class of therapeutic and chemopreventative agents. To understand how ERβ regulates genes, we identified genes regulated by the unliganded and liganded forms of ERα and ERβ in U2OS cells. Microarray data demonstrated that virtually no gene regulation occurred with unliganded ERα, whereas many genes were regulated by estradiol (E2). These results demonstrated that ERα requires a ligand to regulate a single class of genes. In contrast, ERβ regulated three classes of genes. Class I genes were regulated primarily by unliganded ERβ. Class II genes were regulated only with E2, whereas class III genes were regulated by both unliganded ERβ and E2. There were 453 class I genes, 258 class II genes, and 83 class III genes. To explore the mechanism whereby ERβ regulates different classes of genes, chromatin immunoprecipitation-sequencing was performed to identify ERβ binding sites and adjacent transcription factor motifs in regulated genes. AP1 binding sites were more enriched in class I genes, whereas ERE, NFκB1, and SP1 sites were more enriched in class II genes. ERβ bound to all three classes of genes, demonstrating that ERβ binding is not responsible for differential regulation of genes by unliganded and liganded ERβ. The coactivator NCOA2 was differentially recruited to several target genes. Our findings indicate that the unliganded and liganded forms of ERβ regulate three classes of genes by interacting with different transcription factors and coactivators.

Renal antioxidant enzyme mRNA levels are increased in rats with experimental diabetes mellitus

Summary Exposure to high glucose concentrations increases the mRNA levels of oxygen radical scavenging enzymes in cultured endothelial cells, suggesting a compensatory response to increased free radical production. To test the hypothesis that this response also occurs in vivo, Cu,Zn-superoxide dismutase (Cu,Zn-SOD) and catalase mRNA levels, were measured in the kidneys of Sprague-Dawley rats 17 days after intravenous injection of streptozotocin (60 mg/kg body weight) and compared with those of control rats. Diabetic rats were either left untreated or given differing insulin regimens (2, 3–8, 6–10 IU/day) in two different experiments that were designed to achieve varying degrees of metabolic control. Cu,Zn-SOD and catalase mRNA levels were measured by Northern blot hybridization and standardized by 28S ribosomal RNA determination. Renal Cu,Zn-SOD and catalase mRNA levels were significantly greater in untreated diabetic and in low-dose (2 IU/day) insulin-treated rats than in controls. Treatment with a moderate dose (3–8 IU/day) of insulin normalized catalase but not Cu,Zn-SOD mRNA levels. The highest insulin regimen (6–10 IU/day), in addition to achieving complete metabolic control as evidenced by normal growth and plasma glucose levels, normalized both catalase and Cu,Zn-SOD mRNA levels. Thus, in rats with streptozotocin-induced diabetes Cu,Zn-SOD and catalase renal mRNA levels are greater than in normal rats. This difference is prevented by sufficient insulin dosage to normalize plasma glucose and might be due to an increased production of free radicals. [Diabetologia (1997) 40: 23–29]

The tissue renin-angiotensin system in rats with fructose-induced hypertension: overexpression of type 1 angiotensin II receptor in adipose tissue.

Objective Fructose feeding induces hypertension, insulin-resistance and hypertriglyceridemia in Sprague-Dawley rats. The mechanisms of fructose-induced hypertension are as yet unknown. Here we investigate the effects of fructose feeding and of varying salt intake on blood pressure, glucose tolerance, plasma renin activity, and tissue angiotensinogen, renin, and AT1 receptor mRNA levels in this model of hypertension. Design and methods To investigate the role of the renin-angiotensin system in fructose-induced hypertension we measured angiotensinogen, renin and angiotensin II type 1 (AT1) receptor mRNA levels in tissues of Sprague-Dawley rats that were fed either standard rat chow or a diet containing 66% fructose. Results Blood pressure (P <0.05) and triglyceride (P <0.01) levels were significantly greater in the fructosefed animals. Plasma glucose and insulin responses to an oral glucose load were significantly greater (P <0.05) in fructose-fed than control rats. Angiotensinogen mRNA levels in liver and fat, and renin mRNA levels in kidney did not differ between fructose-fed and control animals. Levels of AT1 receptor mRNA were significantly greater in the fat obtained from fructose-fed rats than in that from control rats (P <0.05), but this was not so in the kidney. To determine whether fructose-induced hypertension is dependent on dietary salt content, rats were fed standard rat chow and a fructose-enriched diet with low and high sodium chloride concentrations. Blood pressure increased significantly (P <0.05) only in the fructose-fed rats receiving the high-salt diet. Similarly, increased AT1 receptor mRNA levels were observed only in the fructosefed rats that were maintained on the high-salt diet. Conclusions Fructose feeding induces hypertension in normal- or high-salt fed animals and it is associated with an increased expression of the AT1 receptor in adipose tissue. These findings suggest that AT1 receptors might play a role in the pathophysiology of metabolic and hemodynamic abnormalities induced by fructose feeding.

Drug and Cell Type-Specific Regulation of Genes with Different Classes of Estrogen Receptor β-Selective Agonists

Estrogens produce biological effects by interacting with two estrogen receptors, ERα and ERβ. Drugs that selectively target ERα or ERβ might be safer for conditions that have been traditionally treated with non-selective estrogens. Several synthetic and natural ERβ-selective compounds have been identified. One class of ERβ-selective agonists is represented by ERB-041 (WAY-202041) which binds to ERβ much greater than ERα. A second class of ERβ-selective agonists derived from plants include MF101, nyasol and liquiritigenin that bind similarly to both ERs, but only activate transcription with ERβ. Diarylpropionitrile represents a third class of ERβ-selective compounds because its selectivity is due to a combination of greater binding to ERβ and transcriptional activity. However, it is unclear if these three classes of ERβ-selective compounds produce similar biological activities. The goals of these studies were to determine the relative ERβ selectivity and pattern of gene expression of these three classes of ERβ-selective compounds compared to estradiol (E2), which is a non-selective ER agonist. U2OS cells stably transfected with ERα or ERβ were treated with E2 or the ERβ-selective compounds for 6 h. Microarray data demonstrated that ERB-041, MF101 and liquiritigenin were the most ERβ-selective agonists compared to estradiol, followed by nyasol and then diarylpropionitrile. FRET analysis showed that all compounds induced a similar conformation of ERβ, which is consistent with the finding that most genes regulated by the ERβ-selective compounds were similar to each other and E2. However, there were some classes of genes differentially regulated by the ERβ agonists and E2. Two ERβ-selective compounds, MF101 and liquiritigenin had cell type-specific effects as they regulated different genes in HeLa, Caco-2 and Ishikawa cell lines expressing ERβ. Our gene profiling studies demonstrate that while most of the genes were commonly regulated by ERβ-selective agonists and E2, there were some genes regulated that were distinct from each other and E2, suggesting that different ERβ-selective agonists might produce distinct biological and clinical effects.

Abnormalities of Insulin Receptors in Spontaneously Hypertensive Rats

Insulin resistance is present in some strains of rats with genetic hypertension. To determine whether this abnormality is present at the level of the insulin receptor, we compared insulin sensitivity, insulin receptor binding, and mRNA levels in tissues of 10-week-old spontaneously hypertensive rats (SHR) and their normotensive Wistar-Kyoto (WKY) controls. Because we have previously demonstrated an inverse relationship between dietary sodium intake and renal insulin receptor density and mRNA levels in normal Sprague-Dawley rats, the two rat strains in the current experiment were fed either low salt (0.07% NaCl) or high salt (7.5% NaCl) chow until the SHR became hypertensive. Fasting plasma glucose and plasma insulin levels did not differ between SHR and WKY and were not affected by salt intake. When the rats were maintained on the low salt diet, the rate of glucose infusion required to main euglycemia during a hyperinsulinemic clamp was significantly lower in SHR than WKY. High salt diet decreased the rate of glucose utilization during the hyperinsulinemic clamp in WKY but not SHR. During the low salt diet, insulin infusion decreased sodium excretion in both WKY and SHR. When the rats were maintained on the high salt diet, the antinatriuretic response to insulin was blunted in WKY but not SHR. Both the density and mRNA levels of insulin receptor were comparable in the kidney of WKY and SHR, but only WKY had the previously demonstrated decrease in receptor number and mRNA levels when fed the high salt chow. Hepatic insulin receptor mRNA levels were significantly lower in SHR than WKY fed the low salt diet. High salt diet decreased significantly insulin receptor mRNA levels in the liver of WKY but not of SHR. Thus, SHR appear to have lost the feedback mechanism that normally limits insulin-induced sodium retention when extracellular volume is expanded. A decreased expression of insulin receptor in the liver of SHR provides a possible explanation for the insulin resistance and decreased insulin clearance present in this strain.