Ekatherina Stoyanova

Non-invasive high-frequency vascular ultrasound elastography.

Non-invasive vascular elastography (NIVE) was recently introduced to characterize mechanical properties of superficial arteries. In this paper, the feasibility of NIVE and its applicability in the context of high-frequency ultrasound imaging is investigated. First, experiments were performed in vitro on vessel-mimicking phantoms. Polyvinyl alcohol cryogel was used to create two double-layer vessels with different mechanical properties. In both cases, the stiffness of the inner layer was made softer. Radial stress was applied within the lumen of the phantoms by applying incremental static pressure steps with a column of a flowing mixture of water-glycerol. The vessel phantoms were insonified at 32 MHz with an ultrasound biomicroscope to provide cross-section sequences of radio-frequency (RF) ultrasound data. The Lagrangian speckle model estimator (LSME) was used to assess the two-dimensional-strain tensors, and the composite Von Mises elastograms were computed. A new implementation of the LSME based on the optical flow equations was introduced. Deformation parameters were estimated using an inversion algorithm. For each in vitro experiment, both layers of approximately 1 mm were distinguished. Second, the use of the method for the purpose of studying small vessels (MicroNIVE) in genetically engineered rodents was introduced. Longitudinal scans of the carotid artery were performed at 40 MHz. The in vivo results give confidence in the feasibility of MicroNIVE as a potential tool to non-invasively study the impact of targeted genes on vascular remodelling in rodents.

Mice Overexpressing Both Human Angiotensinogen and Human Renin as a Model of Superimposed Preeclampsia on Chronic Hypertension

Preeclampsia is the major cause of maternal and fetal mortality/morbidity. Because hypertension is an important risk factor for preeclampsia, we investigated whether hypertensive mice that overexpress human renin and angiotensinogen develop superimposed preeclampsia. Given that the mechanisms underlying this disease are still poorly understood, animal models are of great use for elucidatation. Blood pressure and proteinuria were measured by telemetry and ELISA, respectively. Heart function was evaluated by echocardiography, whereas pathological cardiac hypertrophy–related genes were assessed by real-time PCR. Soluble fms-like tyrosine kinase 1 plasma concentrations were quantitated by ELISA and placental expression by real-time PCR. Transgenic mice develop de novo proteinuria during gestation and marked blood pressure elevation, which are hallmarks of superimposed preeclampsia on chronic hypertension. Abnormal placentation present in these mothers produced a significant decrease in pup and placental weight and was associated with an increased placental expression of soluble fms-like tyrosine kinase 1. We also found heightened circulating levels of this receptor, when adjusted for placental mass, as has been observed in women who suffer from preeclampsia. Cardiac hypertrophy could be observed in the transgenic mice and was exacerbated by gestation. As a result, heart function was significantly decreased, and markers of pathological hypertrophy were increased. Our data, thus, confirm the characterization of a new model of superimposed preeclampsia on chronic hypertension. Because chronically hypertensive women are at risk of developing the pathology, our model reflects a clinical reality and is, thus, an excellent tool to elucidate the molecular mechanisms triggering this disease.

Vascular Endothelial Dysfunction in β-Thalassemia Occurs Despite Increased eNOS Expression and Preserved Vascular Smooth Muscle Cell Reactivity to NO

Aims The hereditary β-thalassemia major condition requires regular lifelong blood transfusions. Transfusion-related iron overloading has been associated with the onset of cardiovascular complications, including cardiac dysfunction and vascular anomalies. By using an untransfused murine model of β-thalassemia major, we tested the hypothesis that vascular endothelial dysfunction, alterations of arterial structure and of its mechanical properties would occur despite the absence of treatments. Methods and Results Vascular function and structure were evaluated ex vivo. Compared to the controls, endothelium-dependent vasodilation with acetylcholine was blunted in mesenteric resistance arteries of β-thalassemic mice while the endothelium-independent vasodilator (sodium nitroprusside) produced comparable vessel dilation, indicating endothelial cell impairment with preserved smooth muscle cell reactivity to nitric oxide (NO). While these findings suggest a decrease in NO bioavailability, Western blotting showed heightened expression of aortic endothelial NO synthase (eNOS) in β-thalassemia. Vascular remodeling of the common carotid arteries revealed increased medial elastin content. Under isobaric conditions, the carotid arteries of β-thalassemic mice exhibited decreased wall stress and softening due to structural changes of the vessel wall. Conclusions A complex vasculopathy was identified in untransfused β-thalassemic mice characterized by altered carotid artery structure and endothelial dysfunction of resistance arterioles, likely attributable to reduced NO bioavailability despite enhanced vascular eNOS expression.

Receptor tyrosine kinase Ephb6 regulates vascular smooth muscle contractility and modulates blood pressure in concert with sex hormones.

Background: Eph kinases constitute the largest receptor tyrosine kinase family, and there is no knowledge about their function in blood pressure regulation. Results: Ephb6 regulates vascular smooth muscle cell contraction, and its knock-out resulted in increased blood pressure in castrated male mice. Conclusion: Ephb6 and its ligands can regulate vessel tone and blood pressure. Significance: We have identified a new group of molecules capable of regulating blood pressure. Eph kinases constitute the largest receptor tyrosine kinase family, and their ligands, ephrins (Efns), are also cell surface molecules. Our study is the first to assess the role of Ephb6 in blood pressure (BP) regulation. We observed that EphB6 and all three of its Efnb ligands were expressed on vascular smooth muscle cells (VSMC) in mice. We discovered that small arteries from castrated Ephb6 gene KO males showed increased contractility, RhoA activation, and constitutive myosin light chain phosphorylation ex vivo compared with their WT counterparts. Consistent with this finding, castrated Ephb6 KO mice presented heightened BP compared with castrated WT controls. In vitro experiments in VSMC revealed that cross-linking Efnbs but not Ephb6 resulted in reduced VSMC contractions, suggesting that reverse signaling through Efnbs was responsible for the observed BP phenotype. The reverse signaling was mediated by an adaptor protein Grip1. Additional experiments demonstrated decreased 24-h urine catecholamines in male Ephb6 KO mice, probably as a compensatory feedback mechanism to keep their BP in the normal range. After castration, however, such compensation was abolished in Ephb6 KO mice and was likely the reason why BP increased overtly in these animals. It suggests that Ephb6 has a target in the nervous/endocrine system in addition to VSMC, regulating a testosterone-dependent catecholamine compensatory mechanism. Our study discloses that Ephs and Efns, in concert with testosterone, play a critical role in regulating small artery contractility and BP.

The role of GRIP1 and ephrin B3 in blood pressure control and vascular smooth muscle cell contractility

Several erythropoietin-producing hepatocellular receptor B family (EPHB) and their ligands, ephrinBs (EFNBs), are involved in blood pressure regulation in animal models. We selected 528 single nucleotide polymorphisms (SNPs) within the genes of EPHB6, EFNB2, EFNB3 and GRIP1 in the EPH/EFN signalling system to query the International Blood Pressure Consortium dataset. A SNP within the glutamate receptor interacting protein 1 (GRIP1) gene presented a p-value of 0.000389, approaching the critical p-value of 0.000302, for association with diastolic blood pressure of 60,396 individuals. According to echocardiography, we found that Efnb3 gene knockout mice showed enhanced constriction in the carotid arteries. In vitro studies revealed that in mouse vascular smooth muscle cells, siRNA knockdown of GRIP1, which is in the EFNB3 reverse signalling pathway, resulted in increased contractility of these cells. These data suggest that molecules in the EPHB/EFNB signalling pathways, specifically EFNB3 and GRIP1, are involved blood pressure regulation.

Evidence for a novel mechanism independent of myocardial iron in β-thalassemia cardiac pathogenesis.

Human β-thalassemia major is one of the most prevalent genetic diseases characterized by decrease/absence of β-globin chain production with reduction of erythrocyte number. The main cause of death of treated β-thalassemia major patients with chronic blood transfusion is early cardiac complications that have been attributed to secondary iron overload despite optimal chelation. Herein, we investigated pathophysiological mechanisms of cardiovascular dysfunction in a severe murine model of β-thalassemia from 6 to 15-months of age in the absence of confounding effects related to transfusion. Our longitudinal echocardiography analysis showed that β-thalassemic mice first display a significant increase of cardiac output in response to limited oxygen-carrying erythrocytes that progressed rapidly to left ventricular hypertrophy and structural remodeling. Following this compensated hypertrophy, β-thalassemic mice developed age-dependent deterioration of left ventricular contractility and dysfunction that led toward decompensated heart failure. Consistently, murine β-thalassemic hearts histopathology revealed cardiac remodeling with increased interstitial fibrosis but virtual absence of myocardial iron deposits. Importantly, development of thalassemic cardiac hypertrophy and dysfunction independently of iron overload has uncoupled these cardiopathogenic processes. Altogether our study on β-thalassemia major hemoglobinopathy points to two successive phases resulting from severe chronic anemia and from secondarily induced mechanisms as pathophysiologic contributors to thalassemic cardiopathy.

Comparison of Doppler ultrasound flow resistance indices in beta-thalassemic, sickle cell anemic and control mice

Sickle cell anemia and /spl beta/-thalassemia are two genetic hematological diseases. These conditions trigger thromboembolic events and microcirculatory disorders generating an increase in vascular resistance to blood flow, which can be measured by radiological techniques. The purpose of this study was to demonstrate the possibility of measuring non-invasively peripheral vascular resistance to blood flow in mice using a high-frequency ultrasound imaging system. Four groups were included in the study: 9 control mice, 16 /spl beta/-thalassemic mice (Hbb/sup d-thal/d-thal/) and 9 sickle cell anemic mice (/spl alpha/-/- SAD). The Pourcelot resistive index was used to characterize Doppler waveforms of the carotid artery and abdominal aorta. Resistive indices were significantly increased (p<0.05) in /spl beta/-thalassemic and sickle cell anemic mice compared to controls in both arteries. Measurements of echographic resistive indices allowed detecting the consequences of microrheological blood flow disorders in these small experimental animals. This finding is of high significance in the context of functional genomic studies.