Tgfß/Alk5 signaling is required for shear stress induced klf2 expression in embryonic endothelial cells.

Endothelial cells (EC) translate biomechanical forces into functional and phenotypic responses that play important roles in cardiac development. Specifically, EC in areas of high shear stress, i.e., in the cardiac outflow tract and atrioventricular canal, are characterized by high expression of Krüppel-like factor 2 (Klf2) and by transforming growth factor-beta (Tgfß)-driven endothelial-to-mesenchymal transition. Extraembryonic venous obstruction (venous clip model) results in congenital heart malformations, and venous clip-induced alterations in shear stress-related gene expression are suggestive for an increase in cardiac shear stress. Here, we study the effects of shear stress on Klf2 expression and Tgfß-associated signaling in embryonic EC in vivo using the venous clip model and in vitro by subjecting cultured EC to fluid flow. Cellular responses were assessed by analysis of Klf2, Tgfß ligands, and their downstream signaling targets. Results show that, in embryonic EC, shear stress activates Tgfß/Alk5 signaling and that induction of Klf2 is an Alk5 dependent process.

The endothelin-1 pathway and the development of cardiovascular defects in the haemodynamically challenged chicken embryo.

BACKGROUND/AIMS: Ligating the right lateral vitelline vein of chicken embryos (venous clip) results in cardiovascular malformations. These abnormalities are similar to malformations observed in knockout mice studies of components of the endothelin-1 (ET-1)/endothelin-converting enzyme-1/endothelin-A receptor pathway. In previous studies we demonstrated that cardiac ET-1 expression is decreased 3 h after clipping, and ventricular diastolic filling is disturbed after 2 days. Therefore, we hypothesise that ET-1-related processes are involved in the development of functional and morphological cardiovascular defects after venous clip. METHODS: In this study, ET-1 and endothelin receptor antagonists (BQ-123, BQ-788 and PD145065) were infused into the HH18 embryonic circulation. Immediate haemodynamic effects on the embryonic heart and extra-embryonic vitelline veins were examined by Doppler and micro-particle image velocimetry. Ventricular diastolic filling characteristics were studied at HH24, followed by cardiovascular morphologic investigation (HH35). RESULTS: ET-1 and its receptor antagonists induced haemodynamic effects at HH18. At HH24, a reduced diastolic ventricular passive filling component was demonstrated, which was compensated by an increased active filling component. Thinner ventricular myocardium was shown in 42% of experimental embryos. CONCLUSION: We conclude that cardiovascular malformations after venous clipping arise from a combination of haemodynamic changes and altered gene expression patterns and levels, including those of the endothelin pathway.

In vivo micro particle image velocimetry measurements of blood-plasma in the embryonic avian heart.

The measurement of blood-plasma velocity distributions with spatial and temporal resolution in vivo is inevitable for the determination of shear stress distributions in complex geometries at unsteady flow conditions like in the beating heart. A non-intrusive, whole-field velocity measurement technique is required that is capable of measuring instantaneous flow fields at sub-millimeter scales in highly unsteady flows. Micro particle image velocimetry (muPIV) meets these demands, but requires special consideration and methodologies in order to be utilized for in vivo studies in medical and biological research. We adapt muPIV to measure the blood-plasma velocity in the beating heart of a chicken embryo. In the current work, bio-inert, fluorescent liposomes with a nominal diameter of 400 nm are added to the flow as a tracer. Because of their small dimension and neutral buoyancy the liposomes closely follow the movement of the blood-plasma and allow the determination of the velocity gradient close to the wall. The measurements quantitatively resolve the velocity distribution in the developing ventricle and atrium of the embryo at nine different stages within the cardiac cycle. Up to 400 velocity vectors per measurement give detailed insight into the fluid dynamics of the primitive beating heart. A rapid peristaltic contraction accelerates the flow to peak velocities of 26 mm/s, with the velocity distribution showing a distinct asymmetrical profile in the highly curved section of the outflow tract. In relation to earlier published gene-expression experiments, the results underline the significance of fluid forces for embryonic cardiogenesis. In general, the measurements demonstrate that muPIV has the potential to develop into a general tool for instationary flow conditions in complex flow geometries encountered in cardiovascular research.

Computer aided approximation of flow rate through systemic-pulmonary arterial shunts (SPAS).

The discrimination of flow rates through bronchial arteries that are affected by pathological SPAS today still happens solely qualitatively. A reproducible quantification of flow rates, however, would enable the comprehension of phenomena like the intensified shunt perfusion seen in cases of chronic inflammations or the characterization of SPAS that may cause cardiovascular problems. A computational program is developed, that allows the modeling of individual bronchial arteries on the basis of the information provided by angiography. Angiographic images are available from the standard clinical assessment of SPAS. The flow through continuous and geometrically measurable vessel segments and SPAS is given by the law of Hagen-Poiseuille. The discharge through healthy branches is calculated by means of allometric scaling laws. The simulation results are verified by flow experiments in artificial vessel networks made of glass and PE tubing. The experimental set-up mimics realistic, pulsating pressure and flow conditions. When applied to the artificial vessel networks, the model described herein provides results for the volumetric flow rate that differ from values measured in laboratory experiments by <6%. The computer model is also applied to real angiographic images. Due to inaccuracies during the deduction of the geometry and due to necessary simplifications of the model, we expect significant deviations between calculated and real flow rates in bronchial systems. Nevertheless, the presented method enables the physician to objectively estimate the order of magnitude of volumetric flow through individual SPAS fairly independently from his experience and without the need of measurements additional to the mandatory angiography.