Gab1, SHP2, and protein kinase A are crucial for the activation of the endothelial NO synthase by fluid shear stress

Fluid shear stress enhances NO production in endothelial cells by a mechanism involving the activation of the phosphatidylinositol 3-kinase and the phosphorylation of the endothelial NO synthase (eNOS). We investigated the role of the scaffolding protein Gab1 and the tyrosine phosphatase SHP2 in this signal transduction cascade in cultured and native endothelial cells. Fluid shear stress elicited the phosphorylation and activation of Akt and eNOS as well as the tyrosine phosphorylation of Gab1 and its association with the p85 subunit of phosphatidylinositol 3-kinase and SHP2. Overexpression of a Gab1 mutant lacking the pleckstrin homology domain abrogated the shear stress-induced phosphorylation of Akt but failed to affect the phosphorylation or activity of eNOS. The latter response, however, was sensitive to a protein kinase A (PKA) inhibitor. Mutation of Gab1 Tyr627 to phenylalanine (YF-Gab1) to prevent the binding of SHP2 completely prevented the shear stress-induced phosphorylation of eNOS, leaving the Akt response intact. A dominant-negative SHP2 mutant prevented the activation of PKA and phosphorylation of eNOS without affecting that of Akt. Moreover, shear stress elicited the formation of a signalosome complex including eNOS, Gab1, SHP2 and the catalytic subunit of PKA. In isolated murine carotid arteries, flow-induced vasodilatation was prevented by a PKA inhibitor as well as by overexpression of either the YF-Gab1 or the dominant-negative SHP2 mutant. Thus, the shear stress-induced activation of eNOS depends on Gab1 and SHP2, which, in turn, regulate the phosphorylation and activity of eNOS by a PKA-dependent but Akt-independent mechanism.     

The number of cardiac myocytes in the hypertrophic and hypotrophic left ventricle of the obese and calorie-restricted mouse heart

Changes in body mass due to varying amounts of calorie intake occur frequently with obesity and anorexia/cachexia being at opposite sides of the scale. Here, we tested whether a high-fat diet or calorie restriction (CR) decreases the number of cardiac myocytes and affects their volume. Ten 6–8-week-old mice were randomly assigned to a normal (control group, n = 5) or high-fat diet (obesity group, n = 5) for 28 weeks. Ten 8-week-old mice were randomly assigned to a normal (control group, n = 5) or CR diet (CR group, n = 5) for 7 days. The left ventricles of the hearts were prepared for light and electron microscopy, and analysed by design-based stereology. In CR, neither the number of cardiac myocytes, the relationship between one- and multinucleate myocytes nor their mean volume were significantly different between the groups. In contrast, in the obese mice we observed a significant increase in cell size combined with a lower number of cardiomyocytes (P < 0.05 in the one-sided U-test) and an increase in the mean number of nuclei per myocyte. The mean volume of myofibrils and mitochondria per cardiac myocyte reflected the hypertrophic and hypotrophic remodelling in obesity and CR, respectively, but were only significant in the obese mice, indicating a more profound effect of the obesity protocol than in the CR experiments. Taken together, our data indicate that long-lasting obesity is associated with a loss of cardiomyocytes of the left ventricle, but that short-term CR does not alter the number of cardiomyocytes.     

The suitability of iodinated Angiotensin-(1-7) peptides as pharmacological tools

INTRODUCTION: The heptapeptide Angiotensin-(1-7) [(Ang-(1-7)] is a biologically active component of the Renin-Angiotensin System. Pharmacological studies often involve Ang-(1-7) radioactively labelled with (125)I. Given the small size of the original peptide, we investigated whether introduction of a rather bulky iodine label interferes with the biological activity of Ang-(1-7). METHODS: Ang-(1-7) was labelled with nonradioactive iodine with the chloramine-T method. The reaction products were separated on HPLC and analysed with mass spectrometry. The products were tested for biological activity in two ways: The ability of labelled Ang-(1-7) to block Ang II-induced contraction in rat aortic rings was tested in an organ bath setup. The affinity of labelled angiotensin for ACE in rat plasma was examined in vitro. RESULTS: Iodination of Angiotensin-(1-7) resulted in two main products: monoiodinated and diiodinated Ang-(1-7) that could be easily separated on HPLC. In an organ bath experiment, monoiodinated Ang-(1-7) blocked Ang II responses identical to the native compound, whereas diiodinated Ang-(1-7) had lost its ability to block Ang II responses. Likewise, monoiodinated Ang-(1-7) had retained its affinity for ACE, while the affinity of diiodinated Ang-(1-7) was greatly reduced. DISCUSSION: Monoiodinated Ang-(1-7) has a biological activity identical to the native compound, whereas this is lost in diiodinated Ang-(1-7). Therefore, only the monoiodinated radioactive form seems suited for pharmacological studies.     

Contrast agents in X-ray computed tomography and its applications in oncology

Intravascular iodinated contrast agents are required for a large proportion of computed tomography (CT) studies. Contrast media are indispensable to more clearly differentiate anatomic structures and to detect and characterize abnormalities. Depending on the indication up to 200 ml of these agents are injected during CT. Despite these large amounts adverse effects are rare and have further decreased with the introduction of non-ionic substances. However, it took 10 to 20 years until these non-ionic agents replaced the older ionic agents in clinical practice. In recent years no new substance has been brought to the market. The introduction of rapid scanning using multislice CT technology, however, has led to the development of more sophisticated contrast injection techniques. Current research focuses on optimizing contrast application techniques and on further evaluating the safety profiles of the various substances. The amount of contrast enhancement obtained in individual patients for instance depends on the contrast agent characteristics, such as iodine concentration, and the parameters of the contrast injection protocol, such as iodine flux and iodine dose. Meanwhile, contrast agent characteristics such as osmolality and viscosity play a role in the safety profile of an agent. This paper provides a current overview of CT contrast media, CT contrast dynamics, and CT contrast applications with a special focus on oncological imaging.     

Variability of coronary calcium scores throughout the cardiac cycle: implications for the appropriate use of electrocardiogram-dose modulation with retrospectively gated computed tomography

OBJECTIVE: To study how much the calcium scores at various phases throughout the cardiac cycle deviate from the score in the most motionless phase during retrospectively electrocardiogram (ECG)-gated multidetector row computed tomography (MDCT) of the heart and to evaluate how to optimize ECG-based tube current modulation so that errors in calcium scoring can be minimized while dose savings can be maximized. MATERIALS AND METHODS: In 73 subjects with known or suspected coronary artery disease we performed retrospectively ECG-gated 64-detector row computed tomography for calcium scoring. Four subjects were excluded after scanning because of breathing artifacts or lack of coronary calcification. The scans of 69 subjects (46 men, mean age 62 +/- 6 years) were used for further analysis. Heart rate during the scan was recorded. In each patient, calcium scoring [Agatston score (AS), mass score (MS), and volume score, (VS)] was performed on 10 data sets reconstructed at 10%-intervals throughout the cardiac cycle. The most motionless phase was subjectively determined and used as the reference phase. For the score in each phase, deviation from the score in the reference phase was determined. An ECG-simulator was used to determine the amount of dose saving while scanning with dose modulation and applying diagnostic dose during 1 or several phases. RESULTS: Mean heart rate was 63 (+/-13) beats per minute (bpm). In 51% of patients the reference phase was the 70% phase. Using the calcium score in the 70% phase (mid-diastole) instead of the reference at heart rates below 70 bpm would have induced a median score deviation of 0% [interquartile range: 0%-6% (AS, MS, and VS)] and using the calcium score in the 40% phase (end-systole) at heart rates > or =70 bpm would also have induced a median score deviation of 0% [interquartile range: 0%-7% (AS), 0%-5% (MS), and 0%-3% (VS)]. Errors in calcium scores of more than 10% occur in around 10% of subjects for all 3 scoring algorithms. Dose savings increased with lower heart rates and shorter application of diagnostic dose. CONCLUSIONS: The optimum phases for dose modulation are 70% (mid-diastole) at heart rates below 70 bpm and 40% (end-systole) at heart rates above 70 bpm. Under these conditions dose saving is maximum and a median error of 0% is found for the various calcium scoring techniques with score errors of more than 10% in around 10% of subjects.     

High blood pressure in pregnancy and coronary calcification

A considerable proportion of pregnant women develop high blood pressure in pregnancy. Although it is assumed that this condition subsides after pregnancy, many of these women develop the metabolic syndrome later in life and are at increased risk to develop coronary heart disease. Atherosclerosis development is considered in between risk factors and occurrence of vascular symptoms. We set out to cross-sectionally study the relation of high blood pressure during pregnancy with risk of coronary calcification. The study population was composed 491 healthy postmenopausal women selected from a population-based cohort study. Information on high blood pressure during pregnancy was obtained using a questionnaire. Between 2004 and 2005, the women underwent a multidetector computed tomography (Philips Mx 8000 IDT 16) to assess coronary calcium. The Agatston score, volume, and mass measurements were used to quantify coronary calcium. A total of 30.7% of the women reported to have had high blood pressure in pregnancy. Body mass index (odds ratio [OR]: 1.05; 95% CI: 1.01 to 1.09) and diastolic blood pressure (OR: 1.03; 95% CI: 1.01 to 1.05) were significantly related to a history of high blood pressure in pregnancy. Age was significantly related to increased coronary calcification. Women with a history of high blood pressure during pregnancy had a 57% increased risk of having coronary calcification compared with those women without this condition (OR: 1.57; 95% CI: 1.04 to 2.37). After adjusting for age, the relation did not change (OR: 1.64; 95% CI: 1.07 to 2.53). We concluded that high blood pressure during pregnancy is associated with an increased risk of coronary calcification later in life.