Breast dosimetry using high-resolution voxel phantoms.

A computer model of X-ray mammography has been developed, which uses quasi-realistic high-resolution voxel phantoms to simulate the breast. The phantoms have 400 microm voxels and simulate the three-dimensional distributions of adipose and fibro-glandular tissues, Cooper's ligaments, ducts and skin and allow the estimation of dose to individual tissues. Calculations of the incident air kerma to mean glandular dose conversion factor, g, were made using a Mo/Mo spectrum at 28 kV for eight phantoms in the thickness range 40-80 mm and of varying glandularity. The values differed from standard tabulations used for breast dosimetry by up to 43%, because of the different spatial distribution of glandular tissue within the breast. To study this further, additional voxel phantoms were constructed, which gave variations of between 9 and 59% compared with standard values. For accurate breast dosimetry, it is therefore very important to take the distribution of glandular tissues into account.

Calculation of the properties of digital mammograms using a computer simulation.

A Monte Carlo computer model of mammography has been developed to study and optimise the performance of digital mammographic systems. The program uses high-resolution voxel phantoms to model the breast, which simulate the adipose and fibroglandular tissues, Cooper's ligaments, ducts and skin in three dimensions. The model calculates the dose to each tissue, and also the quantities such as energy imparted to image pixels, noise per image pixel and scatter-to-primary (S/P) ratios. It allows studies of the dependence of image properties on breast structure and on position within the image. The program has been calibrated by calculating and measuring the pixel values and noise for a digital mammographic system. The thicknesses of two components of this system were unknown, and were adjusted to obtain a good agreement between measurement and calculation. The utility of the program is demonstrated with the calculations of the variation of the S/P ratio with and without a grid, and of the image contrast across the image of a 50-mm-thick breast phantom.

Monte Carlo simulation of a mammographic test phantom.

A test phantom, including a wide range of mammographic tissue equivalent materials and test details, was imaged on a digital mammographic system. In order to quantify the effect of scatter on the contrast obtained for the test details, calculations of the scatter-to-primary ratio (S/P) have been made using a Monte Carlo simulation of the digital mammographic imaging chain, grid and test phantom. The results show that the S/P values corresponding to the imaging conditions used were in the range 0.084-0.126. Calculated and measured pixel values in different regions of the image were compared as a validation of the model and showed excellent agreement. The results indicate the potential of Monte Carlo methods in the image quality-patient dose process optimisation, especially in the assessment of imaging conditions not available on standard mammographic units.

Cardiotrophin-1 increases angiotensinogen mRNA in rat cardiac myocytes through STAT3 : an autocrine loop for hypertrophy.

-Cardiotrophin-1, an interleukin-6-related cytokine, stimulates the Janus kinase/signal transducers and activators of transcription (JAK/STAT) pathway and induces cardiac myocyte hypertrophy. In this study, we demonstrate that cardiotrophin-1 induces cardiac myocyte hypertrophy in part by upregulation of a local renin-angiotensin system through the JAK/STAT pathway. We found that cardiotrophin-1 increased angiotensinogen mRNA expression in cardiac myocytes via STAT3 activation. Tyrosine phosphorylation of STAT3 by cardiotrophin-1 treatment resulted in STAT3 homodimer binding to the St-domain in the angiotensinogen gene promoter, which lead to promoter activation in a transient transfection assay. Cardiotrophin-1-induced STAT3 tyrosine phosphorylation and binding to the St-domain were suppressed by AG490, a specific JAK2 inhibitor, which also attenuated cardiotrophin-1-stimulated angiotensinogen promoter activity. Cardiotrophin-1 did not activate the angiotensinogen gene promoter that contained a substitution mutation within the St-domain. Finally, losartan, an angiotensin II type 1 receptor antagonist, significantly attenuated cardiotrophin-1-induced hypertrophy of neonatal rat cardiac myocytes. Angiotensin II is known to induce cardiac myocyte hypertrophy by activating the G-protein-coupled angiotensin II type 1 receptor. Our results suggest that upregulation of angiotensinogen and angiotensin II production contribute to cardiotrophin-1-induced cardiac myocyte hypertrophy and emphasize an important interaction between G-protein-coupled and cytokine receptors.

Angiotensin II-stimulated induction of sis-inducing factor is mediated by pertussis toxin-insensitive G(q) proteins in cardiac myocytes.

The Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway is stimulated by angiotensin II (Ang II) via the type 1 receptor after acute pressure overload in the heart. The purpose of this study was to determine whether activation of the JAK-STAT pathway by Ang II is dependent on G proteins. Ang II (100 nmol/L for 120 minutes) caused formation of sis-inducing factor (SIF) complexes and tyrosine phosphorylation of STAT proteins in neonatal rat ventricular myocytes. The percentage of change in Ang II-stimulated SIF induction was not affected by pertussis toxin (PTX) or GP antagonist-2A, compounds that inhibit activation of G(i) and G(o) proteins. In contrast, GP antagonist-2A, a peptide that selectively inhibits activation of G(q) proteins, completely abolished Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation. Pretreatment of cardiac myocytes with U73122, an inhibitor of phosphatidylinositol-specific phospholipase C (PLC) activity, decreased Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation in a dose-dependent manner. Chelation of intracellular Ca(2+) with BAPTA-AM did not alter Ang II-stimulated SIF induction. In contrast, pretreatment of cardiac myocytes with Ro-31-8220, a potent and specific inhibitor of protein kinase C (PKC), decreased Ang II-stimulated SIF induction in a dose-dependent manner. Ang II-stimulated SIF induction was abolished in cardiac myocytes after downregulation of PKC by treatment with PMA. From these data, we conclude that Ang II-stimulated SIF induction and STAT3 tyrosine phosphorylation is mediated by PTX-insensitive G proteins through a G(q)-PLC-PKC-mediated pathway in neonatal rat ventricular myocytes.

alpha-Thrombin inhibits signal transducers and activators of transcription 3 signaling by interleukin-6, leukemia inhibitory factor, and ciliary neurotrophic factor in CCL39 cells.

We recently demonstrated that, in rat aortic smooth muscle cells, alpha-thrombin stimulated Stat3/SIF-A (signal transducers and activators of transcription 3/sis-inducing factor-A) activity [G. J. Bhat et al. (1997) Hypertension 29(Pt. 2), 356-360]. In the present study, we observed that exposure of CCL39 cells (a Chinese hamster lung fibroblast cell line) to alpha-thrombin resulted in a time-dependent decrease in basal SIF-A activity. We hypothesized that the decrease in basal SIF-A was due to the initiation of an inhibitory pathway, following alpha-thrombin exposure. To test this hypothesis, we determined if alpha-thrombin would inhibit Stat3 and SIF-A activation by interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF). In support of this hypothesis, alpha-thrombin inhibited the Stat3/SIF-A response induced by all the above cytokines. The inhibition by alpha-thrombin was concentration dependent, was sensitive to hirudin, and was mimicked by the thrombin receptor agonist peptide. The inhibition did not require the activation of phorbol 12-myristate 13-acetate-sensitive isoforms of protein kinase C and was reversed by pretreatment with the mitogen-activated protein kinase kinase 1 (MAPKK1 or MEK1) inhibitor PD98059. Inhibitory cross talk between alpha-thrombin and IL-6 was also observed in MRC-5 cells, a fibroblast cell line derived from human lung tissue. Thus, we identify a novel alpha-thrombin inhibitory pathway which, acting through a MAPKK1-dependent mechanism, blocks IL-6-, LIF-, and CNTF-induced Stat3/SIF-A activation. This inhibitory cross talk may provide an important regulatory function to modulate gene transcription by these cytokines, during immune and inflammatory responses.

The type I angiotensin II receptor couples to Stat1 and Stat3 activation through Jak2 kinase in neonatal rat cardiac myocytes.

The octapeptide, angiotensin II, has a modulatory role on cardiac cellular growth associated with hypertension and in compensatory remodeling following myocardial infarction. The molecular signal transduction pathways that participate in these and other cellular actions in response to angiotensin II are presently being elucidated. The signal transducers and activators of transcription (STAT) pathway directly links cytokine and growth factor receptors with transcriptional activity. We provide evidence that the G protein-linked, angiotensin II, AT1-receptor couples to activation of the STAT pathway in neonatal rat cardiac myocytes. Angiotensin II induces primarily sis-inducing factor (SIF) B and to a lesser extent SIF-C and SIF-A. The EC50 of this response was 40 nM and Stat1 and Stat3 proteins were identified as components of the SIF complexes. Stat1 and Stat3 were tyrosine phosphorylated five-fold and three-fold, respectively, over control levels following angiotensin II treatment of cardiac myocytes. Phosphorylation of Stat1 and Stat3 proteins was rapid (5 min) and sustained (60 min). Jak2 was also tyrosine phosphorylated eight-fold by angiotensin II treatment, and phosphorylated Stat1 and Stat3 proteins co-immunoprecipitated with activated Jak2 kinase. Selective inhibition of Jak2 kinase with AG-490 blocked formation of angiotensin II induced SIF complexes, suggesting that Jak2 kinase is required for cardiomyocyte SIF induction. In addition, Jak2, Stat1 and Stat3 proteins co-immunoprecipitated with the AT1-receptor. These are the first data to demonstrate coupling of a G-protein coupled receptor, AT1, to the JAK-STAT pathway in primary cultured cardiac myocytes and suggest that this pathway may be involved in transcriptional regulation by angiotensin II.

Molecular mechanisms of angiotensin II in modulating cardiac function: intracardiac effects and signal transduction pathways.

Angiotensin II (Ang II), the effector peptide of the renin-angiotensin system (RAS), regulates volume and electrolyte homeostasis and is involved in cardiac and vascular cellular growth in humans and other species. This system, which has been conserved throughout evolution, plays an important role in cardiac and vascular pathology associated with hypertension, coronary heart disease, myocarditis and congestive heart failure. The traditional RAS is viewed as a system in which circulating Ang II is delivered to target organs and cells. However, in the past decade, a local RAS has been described in cardiac cells, providing evidence for autocrine and paracrine pathways by which biological actions of Ang II could be mediated. The critical actions of Ang II are mediated primarily through the AT1, G-protein (guanylyl nucleotide binding protein) coupled receptor. In addition to coupling to conventional G-protein signal transduction pathways, the AT1 receptor was recently shown to increase the tyrosine phosphorylation of several intracellular substrates, including the STAT (Signal Transducers and Activators of Transcription) family of novel transcription factors, in rat cardiac fibroblasts, myocytes and vascular smooth muscle cells, and AT1 receptor transfected CHO cells. It has been shown that Ang II stimulates the tyrosine phosphorylation and nuclear translocation of Stat1 (Stat 91) and Stat3 (Stat 92). Angiotensin II acting directly through the AT1 receptor, induces the formation of a complex of STAT proteins termed SIF (sis-inducing factor) which binds the DNA sequence, SIE (sis-inducing element) present in the promotor element of many genes. This provides evidence for a direct role of Ang II in mediating inflammatory and remodeling responses through the JAK-STAT pathway. Thus, it is likely that the JAK-STAT pathway has an important role in Ang II-mediated effects on gene transcription, cardiac and vascular cellular growth/development, and inflammatory responses.

Sympathetic innervation modulates the expression of angiotensin II receptors in embryonic rat heart grafted in oculo.

Angiotensin II acts as a cardiac growth factor, and causes both inotropic and chronotropic changes within the heart. In the present study, we used an in oculo model system to examine the effects of sympathetic innervation on the density of cardiac angiotensin II receptors. Quantitative autoradiography was used to determine the density of angiotensin II receptors in embryonic rat hearts grafted into either sympathetically innervated or sympathetically denervated eye chambers of adult host rats. The density of specific binding to angiotensin II receptors was nearly three-fold higher in sympathetically non-innervated compared to sympathetically innervated heart grafts (30.8 +/- 4.2 v 11.5 +/- 3.2 fmol/mg protein). Specific binding to angiotensin II receptors in heart grafts was displaced by addition of the AT1 receptor antagonist losartan, but not by addition of the AT2 receptor competitor PD 123177. Thus, only AT1 receptors were present in sympathetically innervated and sympathetically non-innervated embryonic rat hearts grafted in oculo. We conclude that changes in sympathetic innervation caused changes in the density of cardiac angiotensin II receptors in the present study. Our results may have implications for growth and function not only during cardiac development, but also during cardiac disease.

Quantification and localisation of angiotensin II receptors and angiotensin converting enzyme in the developing rat heart.

OBJECTIVE: The aim was to determine the quantification and localisation of angiotensin II receptors and angiotensin converting enzyme (ACE) in the developing rat heart. METHODS: Quantitative autoradiography was used to determine the density of [125I]Sar1-angiotensin II binding to angiotensin II receptors and the density of [125I]351A binding to ACE. RESULTS: Angiotensin II receptors were first detected in the myocardium on embryonic day 14, and reached a maximum density within the first postnatal week. During the perinatal period, the density of angiotensin II receptors was twofold higher in atrial than in ventricular myocardium [27(SEM 7) v 11(1) fmol.mg-1 protein]. By adulthood, however, the density of angiotensin II receptors did not differ between atria and ventricles. A high density of angiotensin II receptors was first detected in the cardiac vasculature on embryonic day 19, with binding density decreasing during development. During the embryonic and early postnatal periods, most of the binding in the cardiac vasculature was to AT2 receptors. ACE was first detected in the myocardium on the day of birth, with the density of binding to ACE increasing during development. On postnatal day 56, the density of binding to ACE was fourfold higher in atrial than in ventricular myocardium [99(22) v 22(4) fmol.mg-1 protein]. A moderate density of binding to ACE was first detected in the cardiac vasculature and heart valves on embryonic day 19, with binding density increasing during development. By adulthood, the density of binding to ACE was 10- to 25-fold higher in the cardiac vasculature and heart valves than in the myocardium. CONCLUSIONS: The density of angiotensin II receptors and the density of binding to ACE are developmentally regulated in the heart. Both AT1 and AT2 receptors were present in the rat heart by late gestation and could, therefore, mediate the effects of angiotensin II on early cardiac growth and development.

Developmental sensitivity to high dietary sodium chloride in borderline hypertensive rats.

The present study compared the postweaning blood pressures and body weights of borderline hypertensive rats exposed to a high (8%) sodium chloride maternal diet either from conception to weaning or only during the weaning period with borderline hypertensive rats consistently exposed to a normal (1%) sodium chloride maternal diet. Because the effects of early sodium chloride exposure may be most evident during a subsequent challenge, rats from each group were assigned to receive either an 8% sodium chloride or a 1% sodium chloride diet from 8 to 17 weeks of age. Exposure to an 8% sodium chloride diet from conception through weaning increased the adult blood pressure of borderline hypertensive rats compared with that of controls exposed to a 1% sodium chloride diet; exposure to an 8% sodium chloride diet only during weaning did not increase blood pressure. An 8% sodium chloride diet beginning at 8 weeks of age increased systolic blood pressure. The effects of perinatal and adult exposure to high dietary sodium chloride were additive. Behavioral observations and urinary electrolyte measures confirmed that pups exposed to an 8% sodium chloride diet during weaning ingested the high-sodium chloride diet. The blood pressure and heart rate response to autonomic nervous system ganglionic blockade were assessed at 17 weeks of age. Borderline hypertensive rats exposed to an 8% sodium chloride diet from conception through weaning showed an increased bradycardic response, but no difference in depressor response, to ganglionic blockade. These data suggest that the window of developmental sensitivity for modulation of blood pressure regulation by high dietary sodium chloride occurs during prenatal and early postnatal development.

Effects of long-term air jet noise and dietary sodium chloride in borderline hypertensive rats.

The hypothesis that simultaneous exposure to a high (8%) sodium chloride diet and behavioral stress (air jet noise) would act synergistically to increase blood pressure was investigated in male borderline hypertensive rats. Rats were fed either a 1% or an 8% sodium chloride diet beginning at 6 weeks of age. Rats in the Air Noise condition were restrained and exposed to random blasts of air jet noise for 2 h/d, 5 d/wk, from 7 to 17 weeks of age. Controls either were placed in identical restrainers and test chambers but not exposed to air jet noise (Restrained Control) or were left undisturbed (Maturation Control). Biweekly indirect blood pressure measurements showed that by 17 weeks of age, the high-sodium chloride diet and air jet noise exposure produced additive increases in blood pressure. Direct blood pressure measurements at 18 weeks of age confirmed the higher systolic pressures in borderline hypertensive rats exposed to both an 8% sodium chloride diet and air jet noise. After ganglionic blockade, the blood pressure of rats in the Air Noise group remained higher than that of Restrained and Maturation Controls, suggesting that the increased blood pressure of air jet noise-exposed rats was not maintained by increased autonomic activity. Blood pressure after maximal vasodilation by hydralazine was increased in rats exposed to both an 8% sodium chloride diet and air jet noise compared with other groups. Baroreceptor reflex sensitivity (tested by graded doses of angiotensin II) did not differ among groups.(ABSTRACT TRUNCATED AT 250 WORDS)