Jeannette Fareh

Endothelin-1 and Angiotensin II Receptors in Cells From Rat Hypertrophied Heart: Receptor Regulation and Intracellular Ca2+ Modulation

This study investigates the cellular localization and regulation of endothelin-1 (ET-1) and angiotensin II (Ang II) receptors and the effects of ET-1 and Ang II on [Ca2+]i in cardiac hypertrophy due to volume overload in the rat. Radioligand binding assays and [Ca2+]i measurements by fura 2 methodology were performed on isolated ventricular cardiomyocytes and fibroblasts from the heart of rats with a 4-week aortocaval shunt. In the hypertrophied myocardium, ET-1 and Ang II concentrations were unchanged in ventricles. Ventricular ET-1 receptors had a cell-specific distribution: > 90% of ET receptors in cardiomyocytes are of the ETA subtype, whereas fibroblasts had a nearly equal proportion of the ETA and ETB subtypes. ET-1 receptor densities, affinities, and ET-1-induced [Ca2+]i were not significantly different from control in both ventricular cell types from hypertrophied myocardium. Ang II specific binding was very low on isolated ventricular cardiomyocytes, suggesting few receptors in control conditions. However, [Ca2+]i responses induced by Ang II at concentrations > 10(-8) mol/L were detectable and were significantly higher in hypertrophied cardiomyocytes. Ang II receptor density (exclusively AT1) on fibroblasts was significantly reduced (42,970 +/- 3330 versus 73,870 +/- 7940 sites per cell for control cells, P < .01), but AT1 receptor affinity was unchanged after volume overload. The maximum increase in [Ca2+]i evoked by 10(-6) to 10(-4) mol/L Ang II was significantly lower in fibroblasts from overloaded hearts. In conclusion, ET-1 receptor proportion is cell specific, with cardiomyocytes possessing predominantly the ETA subtype and fibroblasts possessing both ETA and ETB receptors. Plasma and cardiac ET-1 concentrations and ET-1 receptor regulation on both ventricular cell types are not altered in cardiac volume overload, suggesting that cardiac ET-1 may not play a significant role in this model. Cardiac hypertrophy induced a significant downregulation of AT1 receptors on fibroblasts, whereas total binding and [Ca2+]i sensitivity to Ang II were significantly enhanced in hypertrophied cardiomyocytes. This suggests that cardiac Ang II may be involved in the pathophysiology of the cardiac hypertrophy of volume overload.

Cellular Effects of β-Particle Delivery on Vascular Smooth Muscle Cells and Endothelial Cells A Dose-Response Study

Background—Although endovascular radiotherapy inhibits neointimal hyperplasia, the exact cellular alterations induced by β irradiation remain to be elucidated. Methods and Results—We investigated in vitro the ability of 32P-labeled oligonucleotides to alter (1) proliferation of human and porcine vascular smooth muscle cells (VSMCs) and human coronary artery endothelial cells (ECs), (2) cell cycle progression, (3) cell viability and apoptosis, (4) cell migration, and (5) cell phenotype and morphological features. β radiation significantly reduced proliferation of VSMCs (ED50 1.10 Gy) and ECs (ED50 2.15 Gy) in a dose-dependent manner. Exposure to β emission interfered with cell cycle progression, with induction of G0/G1 arrest in VSMCs, without evidence of cell viability alteration, apoptosis, or ultrastructural changes. This strategy also proved to efficiently inhibit VSMC migration by 80% and induce contractile phenotype appearance, as shown by the predominance of α-actin immunostaining in β-irradiated ce...

Cytosolic Calcium Changes Induced by Angiotensin II in Neonatal Rat Atrial and Ventricular Cardiomyocytes Are Mediated via Angiotensin II Subtype 1 Receptors

We determined the effects of angiotensin II (Ang II) on cytosolic free calcium concentrations ([Ca2+]i) in the absence and presence of the selective angiotensin subtype 1 (AT1) receptor antagonist losartan or the selective AT2 antagonist PD 123319 in cultured neonatal rat atrial and ventricular cardiomyocytes. We also Ang II receptor density, affinity, and mRNA expression. [Ca2+]i was measured in single cells microphotometrically and by fluorescent digital imaging with fura 2 methodology. Receptor parameters were assessed by competitive binding studies with 125I-[Sar1,Ile8]Ang II in the presence of increasing concentrations of [Sar1,Ile8]Ang II, losartan, and PD 123319. AT1 receptor (types AT1A and AT1B) mRNA abundance was measured by reverse transcription-polymerase chain reaction. Ang II produced concentration-dependent increases in [Ca2+]i values in atrial and ventricular cells were similar but Ang II (10-9 mol/L)-induced [Ca2+]i changes were significantly greater in atrial compared with ventricular cells Ang II responses were blocked by losartan (10-7 mol/L) but not PD 123319 (10-7 mol/L). Binding studies demonstrated a single class of high-affinity. Ang II binding sites on cardiomyocyte membranes (Kd = 0.71 +/- 0.11 mumol/L). 125I-[Sar1,Ile8]Ang II was displaced by losartan but not by PD 123319. AT1 receptor mRNA was detected by reverse transcription-polymerase chain reaction in cells from atria and ventricles. In atrial cardiomyocytes, both AT1A and AT1B receptor genes were expressed, whereas in ventricular cardiomyocytes, only the AT1A receptor gene was expressed. These data demonstrate that neonatal cardiomyocytes possess Ang II receptors of the AT1 receptor subtype that are linked to [Ca2+]i signaling pathways. The different Ang II-induced [Ca2+]i responses between atrial and ventricular cells may be related to differences in the distribution of AT1 receptor subtype subvariants.

Effect of ionizing radiation on thymidine uptake, differentiation, and VEGFR2 receptor expression in endothelial cells: The role of VEGF165

Purpose: Late thrombosis of irradiated vascular segments may be the consequence of endothelial cell (EC) dysfunction after radiation therapy. We investigated the effects of β ionizing radiation on human EC viability, thymidine uptake, and differentiation. Methods and Materials: Endothelial cells were exposed to 32P-labeled DNA oligonucleotides in incremental doses of 2, 6, and 10 Gy. The modulation of the VEGFR2 receptor expression after irradiation and the overall potential radioprotective effect of VEGF165 on these functions were assayed. Results: A dose-dependent inhibitory effect of β irradiation on ECs’ thymidine uptake and differentiation was observed. EC viability, however, was not affected at levels of radiation up to 10 Gy. VEGF165 proved to have a radioprotective effect as ECs’ thymidine uptake, after radiation doses of 2, 6, and 10 Gy, was increased by 1.5-, 2-, and 4-fold, respectively, in the presence of 10 ng/ml of VEGF165 (p < 0.05 vs. LacZ). This concentration of VEGF165 also proved beneficial in maintaining cell differentiation at 16 h postirradiation when compared to controls. These biologic effects were in direct correlation with the upregulation of VEGFR2 receptor expression in irradiated ECs. Conclusions: β irradiation interacts directly with EC functions by significantly reducing their ability to differentiate and proliferate, associated with upregulation of VEGFR2. These effects can be prevented in part by pretreating cells with VEGF165, an effect potentially favored by the upregulation of VEGFR2 receptor expression after irradiation.

Intracellular Ca2+ Modulation by Angiotensin II and Endothelin-1 in Cardiomyocytes and Fibroblasts From Hypertrophied Hearts of Spontaneously Hypertensive Rats

The vasoactive peptides angiotensin II (Ang II) and endothelin-1 (ET-1) have been implicated in cardiac hypertrophy. This study investigates Ang II and ET-1 effects on intracellular free calcium concentration and the receptor subtype through which agonist-induced calcium responses are mediated in isolated cardiomyocytes and fibroblasts from hypertrophied hearts of spontaneously hypertensive rats (SHR). We measured intracellular free calcium concentration by fura 2 methodology and determined receptor status by radioligand binding assays. Ang II (10(-12) to 10(-7) mol/L) had no effect on cardiomyocyte calcium levels in control Wistar-Kyoto rats but significantly increased (P < .01) intracellular free calcium concentration in a dose-dependent manner in cardiomyocytes from SHR. Ang II total and specific binding were increased (P < .05) in SHR cardiomyocytes. Calcium responses elicited by 10(-7) to 10(-5) mol/L Ang II were significantly reduced (P < .01) in SHR fibroblasts despite no significant change in Ang II receptor density. The angiotensin type 1 receptor blocker losartan (1 mumol/L) blocked Ang II-stimulated calcium transients, whereas the angiotensin type 2 receptor blocker PD 123319 had no effect. ET-1- and sarafotoxin S6c-induced calcium responses in cardiomyocytes and fibroblasts were not different between hypertensive and control groups. In conclusion, Ang II and ET-1 elicit distinct and differential responses in a cell-specific manner in cardiomyocytes and fibroblasts from hypertrophied hearts of SHR. Whereas Ang II-mediated effects, which are elicited via angiotensin type 1 receptors, are detectable in cardiomyocytes from SHR, responses to Ang II are blunted in fibroblasts from SHR, and ET-1-related actions are similar in cells from both rat groups. Stimulation of cardiomyocytes by Ang II in hypertrophied hearts associated with pressure overload in genetic hypertension suggests that Ang II could modulate the function of cardiomyocytes of SHR but not those of Wistar-Kyoto rats, whereas cardiac actions of ET-1 do not change with the development of hypertension.

Circulating Osteoglycin and NGAL/MMP9 Complex Concentrations Predict 1-Year Major Adverse Cardiovascular Events After Coronary Angiography

Objective—Previous proteomics experiments have demonstrated that several proteins are differentially expressed in vulnerable human carotid plaques compared with stable plaques. This study aims to investigate the prognostic value of 13 such circulating biomarkers in patients with coronary artery disease. Approach and Results—Between 2008 and 2011, 768 patients who underwent coronary angiography for acute coronary syndrome or stable angina pectoris were included in a prospective biomarker study. Plasma concentrations of 13 biomarkers were measured in 88 patients who experienced a major adverse cardiovascular event (MACE) within 1 year and 176 control patients without MACE who were matched on age, sex, and number of diseased coronary vessels. MACE comprised all-cause mortality, acute coronary syndrome, unplanned coronary revascularization, and stroke. After adjustment for established cardiovascular risk factors, osteoglycin (OGN; odds ratio per SD increase in ln-transformed OGN, 1.53; 95% confidence interval, 1.11–2.11; P=0.010) and neutrophil gelatinase–associated lipocalin/matrix metalloproteinase 9 (NGAL/MMP9; odds ratio per SD increase in ln-transformed NGAL/MMP9, 1.37; 95% confidence interval, 1.01–1.85; P=0.042) complex were independently associated with MACE during follow-up. These associations were independent of C-reactive protein levels. Adding OGN or NGAL/MMP9 to a model containing conventional risk factors did not significantly improve discriminatory power (OGN: area under receiver operating characteristic curve, 0.75 versus 0.67; NGAL/MMP9: 0.73 versus 0.67) but did significantly improve risk reclassification (OGN: net reclassification index=0.29; 95% confidence interval, 0.05–0.53; P<0.019; NGAL/MMP9: net reclassification index=0.44; 95% confidence interval, 0.20–0.69; P<0.001). Conclusions—Circulating OGN and NGAL/MMP9 complex are promising biomarkers that are expressed in vulnerable atherosclerotic plaques and may have incremental value for prediction of MACE within 1 year after coronary angiography.

Cardiac Type-1 Angiotensin II Receptor Status in Deoxycorticosterone Acetate–Salt Hypertension in Rats

The regulation of angiotensin II (Ang II) receptors and Ang II-induced modulation of intracellular Ca2+ concentration in cardiac cells from hearts of experimentally induced hypertensive deoxycorticosterone acetate (DOCA)-salt and control unilaterally nephrectomized (Uni-Nx) Sprague-Dawley rats was assessed. Ang II receptor density and intracellular Ca2+ concentration measurements were examined in adult ventricular myocytes and fibroblasts by radioligand binding assay and digital imaging using fura 2 methodology, respectively. Four-week DOCA-salt treatment induced hypertension associated with cardiac hypertrophy. Ang II binding studies demonstrated that adult ventricular myocytes and fibroblasts possess mainly the AT1 subtype receptor. Moreover, DOCA-salt hypertension was associated with a 1.8-fold increase in Ang II-specific binding compared with myocytes from Uni-Nx control rats. Intracellular Ca2+ responses induced by increasing Ang II concentrations (10[-12] to 10[-4] mol/L) were significantly enhanced in cardiomyocytes from DOCA-salt rats. The effects of Ang II on intracellular Ca2+ spike frequency were unaltered in cardiomyocytes from DOCA-salt-hypertensive rats. The density of AT1 subtype receptors was not modified in ventricular fibroblasts after DOCA-salt treatment. Ang II increased intracellular Ca2+ concentration similarly in ventricular fibroblasts from normal and hypertensive rats. In conclusion, DOCA-salt hypertension is characterized by an increased AT1 receptor density and intracellular calcium responses in ventricular myocytes, whereas in ventricular fibroblasts the AT1 receptor status is unaltered. These findings report for the first time the cardiac cell-specific implication of Ang II and the intracellular calcium signaling pathway stimulated by the AT1 receptor in cardiac hypertrophy in DOCA-salt-hypertensive rats.

Basal and Angiotensin II–Induced Cytosolic Free Calcium in Adult Rat Cardiomyocytes and Fibroblasts After Volume Overload

This study investigates basal and angiotensin II (Ang II)-induced [Ca2+]i concentrations in cells from hearts of rats that have undergone cardiac hypertrophy due to volume overload. [Ca2+]i measurements assessed by digital imaging using fura 2 methodology were performed on isolated ventricular cardiomyocytes and fibroblasts from adult rat hearts with a 4-week aortocaval shunt. Long-term aortocaval shunt induced a significant increase in atrial (72%) and ventricular (41%) weights and a large elevation in plasma atrial natriuretic peptide-(1-98) concentration (160%). For adult cardiomyocytes [Ca2+]i measurements are reported as diastolic (average of the lowest points) and systolic intracellular Ca2+ values (average of the maximum points corresponding to the diastolic points) over a 30-second time interval. Basal diastolic [Ca2+]i (99 +/- 4.1 nmol/L for experimental cells versus 90 +/- 4.8 for control cells) was not altered, whereas basal systolic [Ca2+]i was significantly greater in ventricular cardiomyocytes from overload hearts (155 +/- 2.3 versus 129 +/- 4.4 nmol/L for control cells, P < .05). Ang II increased intracellular Ca2+ spike frequency in a concentration-dependent manner in cardiomyocytes from control and overload myocardium. Basal and Ang II-induced intracellular Ca2+ spike frequencies were not modified in cardiomyocytes from hypertrophied hearts. Basal [Ca2+]i in ventricular fibroblasts from overload myocardium was significantly increased (128 +/- 5.1 nmol/L for fibroblasts from hypertrophied hearts versus 104 +/- 3.5 for control cells, P < .05). Ang II-induced [Ca2+]i was lower in fibroblasts from overload myocardium (P < .05).(ABSTRACT TRUNCATED AT 250 WORDS)

Cerebrospinal Aβ11-x and 17-x levels as indicators of mild cognitive impairment and patients’ stratification in Alzheimer’s disease

In the present work, the concentrations of Aβ11-x and Aβ17-x peptides (x=40 or 42), which result from the combined cleavages of β-amyloid precursor protein (AβPP) by β’/α or α/γ-secretases, respectively, were assessed in cerebrospinal fluid (CSF) samples from patients with Alzheimer’s disease (AD) or mild cognitive impairment (MCI). Specific multiplexed assays were set up using new anti-40 and anti-42 monoclonal antibodies (mAbs) for the capture of these N-truncated Aβ peptides and anti-11 or anti-17 mAbs for their detection. The specificity, sensitivity and reproducibility of such assays were assessed using synthetic peptides and human cell models. Aβ11-x and Aβ17-x were then measured in CSF samples from patients with AD (n=23), MCI (n=23) and controls with normal cognition (n=21). Aβ11-x levels were significantly lower in patients with MCI than in controls. Compared with the combined quantification of Aβ1-42, total Tau (T-Tau) and phosphorylated Tau (P-Tau; AlzBio3, Innogenetics), the association of Aβ11-40, Aβ17-40 and T-Tau improved the discrimination between MCI and controls. Furthermore, when patients with MCI were classified into two subgroups (MCI ⩽1.5 or ⩾2 based on their CDR-SB (Cognitive Dementia Rating–Sum of Boxes) score), the CSF Aβ17-40/Aβ11-40 ratio was significantly higher in patients with CDR-SB ⩽1.5 than in controls, whereas neither Aβ1-42, T-Tau nor P-Tau allowed the detection of this subpopulation. These results need to be confirmed in a larger clinical prospective cohort.

Endothelin-1 signaling is altered in cardiac cells from deoxycorticosterone acetate-salt hypertensive rats

The cardiac cellular effects of endothelin-1 (ET-1) on intracellular free Ca2+ concentration ([Ca2+]i) were investigated in deoxycorticosterone acetate (DOCA)-salt rats with severe cardiac hypertrophy. [Ca2+]i was measured by fura-2 methodology in ventricular cardiomyocytes and fibroblasts of DOCA-salt hypertensive and control unilaterally nephrectomized rats (Uni-Nx). Blood pressure and heart weight were increased (p < 0.01) in DOCA-salt rats compared to control rats. ET-1 (10(-12)-10(-6) M) increased [Ca2+]i in a dose-dependent manner in both cell types from control and hypertensive rats. However, ET-1-induced [Ca2+]i responses were significantly attenuated (p < 0.01) in cardiomyocytes and fibroblasts of DOCA-salt rats. Sarafotoxin S6c (S6c) increased [Ca2+]i in fibroblasts but not in cardiomyocytes. In conclusion, ET-1 dose-dependently increased [Ca2+]i in cardiomyocytes (primarily via ETA receptors) and fibroblasts (via ETA and ETB receptors). Cardiac cell ET-1 signaling pathways are blunted in DOCA-salt hypertensive rats. ET-1 may not play a critical role in the pathophysiology of the severe concentric cardiac hypertrophy present in DOCA-salt hypertensive rats.